ToLCNDV resistant melon plants

ABSTRACT

The application concerns melon plants ( Cucumis melo ) resistant to infection with tomato leaf curl New Dehli virus (ToLCNDV). The resistant melon plants have a genomic introgression fragment on chromosome 5 which confers tolerance to ToLCNDV in a dominant manner. Also disclosed are markers for identifying those fragments, methods for identifying or producing resistant melon plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 National Phase of PCT ApplicationNo. PCT/EP2017/067078, filed on Jul. 7, 2017, which claims priority toEP Application No. 16179134.8, filed Jul. 12, 2016, and U.S. PatentApplication No. 62/500,941, filed May 3, 2017, the disclosures of whichare hereby incorporated by reference in their entireties.

The application concerns melon plants (Cucumis melo) resistant toinfection with tomato leaf curl New Dehli virus (ToLCNDV). The resistantmelon plants have a genomic introgression fragment on chromosome 5 whichconfers tolerance to ToLCNDV in a dominant manner. Also disclosed aremarkers for identifying those fragments, methods for identifying orproducing resistant melon plants.

Tomato leaf curl New Dehli virus (ToLCNDV) is classified as Begomovirusbelonging to the family Geminiviridae. ToLCNDV has a bipartite genomeconsisting of two single stranded DNA molecules referred to as DNA A andDNA B (Saez et al., 2016, Annals of Applied Biology).

ToLCNDV was initially found to infect tomato (Solarium lycopersicum)plants in 1995 in India. Later ToLCNDV was found to infect also plantsof other Solanacea species, like Solanum melongena (aubergine), chilipepper (Capsicum spp.) and Solanum tuberosum (potato). In 2012 infectionof curcubit species (courgette, Cucurbita pepo var. giromantiina) byToLCNDV was found in Spain and in 2015 the virus was identified as thedisease source in melon, encumber and courgette in Tunisia. In themeantime, infection of many Curcubitacea species such as Benincasahispida (wax gourd), Citrullus lanatus (watermelon), Cucumis melo(melon), Cucumis melo var. flexuosus (snake melon), Cucumis sativus(cucumber), Cucurbita moschata (musky gourd), Cucurbita pepo (pumpkin),Cucurbita pepo var. giromontiina (courgette), Lagenaria siceraria(bottle gourd), Luffa cylindrica (sponge gourd), Momordica charantia(bitter gourd) have been proven. Infection of weeds (e.g. Ecliptaprostrata—Asteraceae) and other crops such as Hibiscus cannabinus(kenaf—Malvaceae) and Carica papaya (papaya—Caricaceae) was alsoreported. In the Mediterranean region the disease does occur in variouscrop species in Italy (Sicilia), Spain and Tunisia. In Asian countries,infection was proven in different crops in Bangladesh, India, Indonesia,Pakistan, Philippines, Sri Lanka, Taiwan and Thailand. Furtherinformation on geographical distribution of ToLCNDV is lacking, but fromthe observations made today, the virus clearly seems to further spreadgeographically as well as to other crops. Disease symptoms in generalcomprise phenotypic appearance of yellow mosaic on leaves, leaf curling,vein swelling, and plant stunting. Cucurbits upon infection of youngplants with ToLCNDV show stunted growth and decreased or suppressedfruit production. Also fruits showing skin roughness and longitudinalcracking have been reported. Thus, ToLCNDV causes economic losses invarious important crop species and is a major threat, infection ofplants by ToLCNDV occurs persistently by transmission of the virus bythe phloem sucking whitefly (Bemisia tabaci). (European andMediterranean Plant Protection Organization, EPPO RS 2015/114, 2016/024,2016/040, Entry date June 2015).

In sponge gourd resistance to ToLCNDV has been shown to be controlled bya single dominant gene (Islam et al., 2010, Euphytica 174 (1):83-89).

In tomato, transgenic plants resistant to ToLCNDV have been produced bysilencing virus genes (Varma & Praveen, 2006, ISB News Report).

Assays for transmission, of ToLCNDV by mechanically transferring the sapof an infected zucchini plant to non-infected plants from other cucurbitgenera (Cucumis, Cucurbita, Citrullus, Lagenaria) have been developed.Five Cucumis melo accessions (subsp. agrestis var. momordica:Mom-Khalnd/Kharbuja, Mom-PI124Ind/PI 24112, Mom-PI124Ind/PI 414723 andsubsp. agrestis wild types: Ag-WM9Ind/WM9, Ag-WM7Ind/WM7) resistant toToLCNDV were identified (Lopez et al., 2015, Euphytica 204 (3),679-691). Confirmation of resistance of these accessions by using thenatural whitefly infection system, was not performed.

Saez et al. 2016, pages 214-216 (Proceedings of Cucurbitaeceae 2016, theXIth EUCARPIA Meeting on Genetics and Breeding of Cucurbitaceae Jul.24-28, 2016, Warsaw, Poland) describe that ToLCNDV resistance of the C.melo subsp. agrestis accession WM-7 segregated in an F2 population in a3:1 ratio of tolerant:susceptible, suggesting a single dominantresistance gene being present in WM-7. As in Lopez et al. 2015, supra,mechanical inoculation was used, and not natural infection via whiteflyvector.

Various Cucurbita species (C. pepo, C. moschata, C. maxima, C.fraternal, C. ficifolia) have been screened for resistance to ToLCNDV bythe mechanical sap transmission screening assay. Four Curcubita moschataaccessions (PI 604506, PI 381814, Nigerian local, Kurokawa) were foundto show low symptoms upon, mechanical infection with ToLCNDV. Thisresult however could be reproduced upon whitefly infection only for twoof the accession (PI 604506, PI 381834), demonstrating that resistanceto ToLCNDV should be tested not only under artificial conditions but inaddition by using the naturally occurring whitefly infection system(Saez et al., 2016, Annals of Applied Biology). Therefore, it is unclearif the accessions identified to be resistant to infection by ToLCNDV areresistant under natural growing conditions when the virus is transferredby whiteflies.

Attempts to control ToLCNDV infection of crop plants comprise vector(whitefly) control by insecticides applications and adaption of culturalpractices, including use of virus free crop material (transplants),establishment of crop free periods, weed management (eliminating virusinfected weeds) and destruction of infected plants in the field.However, because of the complex epidemiological factors associated withthe disease, these attempts are not always effective (Saez et al., 2016,Annals of Applied Biology).

Therefore, there is a need to establish further measurements forreducing ToLCNDV infections, further spreading of the virus to othergeographical areas and spreading to other crop species. Breeding ofvarieties resistant to ToLCNDV would be essential for managing thedisease.

An object of the present invention is to provide measurements for thecontrol of ToLCNDV infection in melon plants.

The present invention discloses melon plant cells and melon plants beingresistant to infections by TolCNDV.

It is commonly known that ToLCNDV does infect various different plantspecies of the Curcubitaceae species, including melon species. It isalso well known that ToLCNDV is transmitted persistently from infectedplants to non-infected plants by the plant sucking pest Bemisia tabaci(whitefly). Transfer of ToLCNDV from one crop species to different cropspecies or even from weed species to crop species has been demonstrated.Whiteflies may pick up ToLCNDV from outside the controlled area evenfrom different species and transfer it to melon plants grown in thecontrolled area. Whitefly vector control therefore is of limitedeffectiveness for preventing ToLCNDV infection. TolCNDV resistant melonplants have the advantage that they would withstand infection withTolCNDV without major yield losses, even if plants around the area wherethe melon plants are grown are infected with ToLCNDV.

The inventors found a ToLCNDV resistant donor accession of melon andhave mapped the resistance in a segregating population, using whiteflyinfection with a Spanish ToLCNDV strain for resistance phenotyping. AQuantitative Trait Locus (QTL) was identified on chromosome 5 (QTL5) andintrogressed from the wild donor accession into an elite cultivatedmelon line. Seeds of the elite line, comprising the QTL5 introgressionin homozygous form, were deposited by Nunhems B. V. under Accessionnumber NCIMB 42585 in accordance with the Budapest Treaty. In theseseeds the donor genotype for the Single Nucleotide Polymorphism (SNP)markers (SNP_01 to SNP_06) provided herein is present. The donor itselfis not uniform and has white fruit flesh with a low brix. It is not anaccession of agronomic value. The donor is also not available anymore atthe US seed bank. By identifying and transferring QTL5 from the donorinto cultivated melon, it is now possible to make cultivated melonvarieties and cultivars of high agronomic value (with uniformcharacteristics and marketable fruits having high brix and good shelflife) with resistance against ToLCNDV and thus it is possible tocultivate those melon varieties in ToLCNDV infested areas without yieldloss.

“Melon plant cells” or “melon plants” also designated as muskmelon plantcells or muskmelon plants in the art shall be understood in context withthe present invention to be plant cells originating from the speciesCucumis melo or to be plants belonging to the species Cucumis melo.

Cucumis melo, can be classified into: C. melo cantalupensis, C. meloinodorous and C. melo reticulatus. C. melo cantalupensis are alsoreferred to as Cantaloupes and are primarily round in shape withprominent ribs and almost no netting. Most have orange, sweet flesh andthey are usually very fragrant. In contrast to the European cantaloupe,the North American ‘Cantaloupe’ is not of this type, but belongs to thetrue muskmelons. C. melo inodorous (or winter melons) can be subdividedinto different types, such as Honeydew melon, Piel de Sapo, Sugar melon,Japanese melon, etc. C. melo reticulatus is the true muskmelon, withreticulated skin (netted) and includes Galia melons, Sharlyn melons andthe North American cantaloupe.

Melon and the wild relatives of melon, is/are diploid and has have 12pairs of homologous chromosomes, numbered 1 to 12.

“Resistant” or “being resistant to” shall be understood in context ofthe present invention to mean a plant which is a host species of aparticular pathogen and can therefore be infected by a given pathogen,but wherein the plant comprises a genetic element (e.g., anintrogression fragment) resulting in reduction of pathogen growth and/orspreading in the plant after infection compared to the susceptible plantlacking the genetic element. In context of the present invention“resistant” or “being resistant to” in particular refers to plant cellsor plants being resistant to ToLCNDV. Resistance is a relative termwhich can span a range of (different) reactions in the plant cell orplant, triggered by pathogen infection. The effect of those reactions bythe plant cell or plant can be measured by various means. Typically theeffect is measured by defining a symptom level appearing in the plantcell or plant. Typically average symptoms of several plants of a line(e.g. 10 or more) are compared to average symptoms of several plants ofa control line or variety, preferably a susceptible control line orvariety. Thus at least 10 or more individual plants of a line or varietyare scored at one time point and the average disease score iscalculated.

Concerning the present invention, the following commonly known symptomlevels are applied according to phenotypic observations taken afterToLCNDV infection:

1=Dead plant

2=Severe mosaic and curling, chlorosis and growth reduction. No recovery

3=Strong mosaic and curling, chlorosis and growth reduction. No recovery

4=Curling and mosaic, chlorosis, no or mild growth reduction. Norecovery

5=Curling and mosaic, chlorosis, no growth reduction. Slight recovery ofthe upper plant zone

6=Mild curling, mosaic and chlorosis, no growth reduction. Recovery ofthe upper middle plant

7=Mild curling, mosaic and chlorosis, no growth reduction. Symptomsappear only in the lower plant zone

8=Faint mosaic

9=No symptoms

For determining the symptom level (or disease score) preferably youngplants are infected wish ToLCNDV. Young plants are preferably plantshaving the age of the first true leaf being expanded, preferablyapproximately 12-15 days after sowing. Infection is preferably carriedout via feeding of the vector (Bemisia) carrying the virus. For thispurpose plants are germinated and grown under optimal or close tooptimal conditions. The symptom level is preferably determined at leastonce, e.g. 30 days after infection (or later, e.g. 31, 32, 33, 34, 35days after infection). Optionally symptom level is determined twice oreven three times at different time-points following infection to confirmthe result, e.g. a first scoring at approximately 15, 20 or 25 daysafter infection and a second scoring at approximately 30 days afterinfection (or later, e.g. 31, 32, 33, 34, 35 days after infection) withToLCNDV. See also the Examples. In one aspect a plant line is said to beresistant against TolCNDV infection if it has an average disease scoreof 5.0 or higher, while the susceptible control line or variety, such asvariety Gandalf (or Gandalf F1, Nunhems B. V.) or Vedantrais, has anaverage disease score of 2.5 or less, or 2.0 or less, when grown underthe same conditions and infected in the same way.

It has been observed that introgression of a specific fragment locatedon chromosome 5 in a wild melon donor plant into cultivated melon plantsconfers resistance to ToLCNDV infection in cultivated melon plants orcells derived therefrom. It is sufficient that the respective fragmentis present only in the heterozygous state for conferring ToLCNDVresistance, demonstrating that the fragment confers resistance toTolCNDV infection in a dominant manner. Single Nucleotide Polymorphisms(SNPs) on chromosome 5 were identified which are closely linked to thefragment of chromosome 5 conferring ToLCNDV resistance. The SNPnucleotide of the resistant donor (i.e. the nucleotide of theintrogression fragment) is present in homozygous forms in the depositedseeds, i.e. the donor nucleotide is present in homozygous form forSNP_01 to SNP_06 (linked to QTL5). The SNPs can, therefore, be used totest the presence of the introgression fragment comprising the QTL5 in aplant cell, plant tissue, plant part, and/or in marker assistedselection (MAS) to transfer the QTLs into other elite melon lines orvarieties. The SNPs can also be used to select plants comprising smallerintrogressions fragments than the fragments present in the depositedseeds, whereby the smaller sub-fragments retain the QTL. Alternativelythe SNPs can be used to identify other donors which comprise QTL5 and tointrogress these QTLs into cultivated melon.

The present invention therefore relates to melon plant cells or melonplants comprising an introgression fragment from chromosome 5 of aToLCNDV resistant donor plant, wherein the introgression fragmentcomprises the sequence of the donor plant in-between SNP_01 and SNP_06,preferably the sequence of the donor plant in-between SNP_01 and SNP_05,more preferably the sequence of the donor plant in-between SNP_01 andSNP_04. The ToLCNDV resistance conferring QTL is present on theintrogression fragment, as can be determined by a resistance assay asdescribed herein.

In a preferred embodiment the present invention relates to cultivatedmelon plant cells or melon plants comprising an introgression fragmentfrom chromosome 5 of a ToLCNDV resistant donor plant, wherein theintrogression fragment comprises the sequence of the donor plantin-between SNP_02 and SNP_06, preferably the sequence of the donor plantin-between SNP_02 and SNP_05, more preferably the sequence of the donormelon plant in-between SNP_02 and SNP_04.

The present invention further relates to melon plant cells or melonplants comprising an introgression fragment from chromosome 5 of aToLCNDV resistant donor plant, wherein the introgression fragmentcomprises the sequence of the ToLCNDV resistant donor plant in-betweenSNP_03 and SNP_06, preferably the sequence of the ToLCNDV resistantdonor plant in-between SNP_03 and SNP_05.

Most preferred, the present invention relates to melon plant cells ormelon plants comprising an introgression fragment from chromosome 5 of aToLCNDV resistant donor plant, wherein the introgression fragmentcomprises the sequence of the ToLCNDV resistant donor melon plantin-between SNP_03 and SNP_04.

When referring herein to the introgression fragment comprising thesequence “in-between” two SNPs (Single Nucleotide Polymorphisms), thisencompasses in one aspect that one or both of the two SNPs themselvesare also from the resistant donor, i.e. have the donor nucleotide at theSNP position. In another aspect, the two SNPs are from the recipient,e.g. the susceptible melon plant, while only a region between the twoSNPs is from the resistant donor and confers TolCNDV resistance, i.e.the resistance conferring donor fragment lies in-between the two SNPmarkers. So for example, a plant may comprise the introgression fragmentcomposing the sequence of the ToLCNDV resistant donor melon plantin-between SNP_03 and SNP_04, this plant in one aspect comprises aThymine (T) at nucleotide 68 of SEQ ID NO: 3 and/or an Adenine (A) atnucleotide 227 of SEQ ID NO: 4, i.e. the donor nucleotides. In anotheraspect only a region (the whole region or a part thereof) between thesetwo SNPs is from the donor, while SNP_03 and SNP_04 are from therecipient, having e.g. a Cytosine (C) at nucleotide 68 of SEQ ID NO: 3and e.g. a Guanine (G) at nucleotide 227 of SEQ ID NO: 4.

Thus, regarding the QTL on chromosome 5, SNP_01, SNP_02, SNP_03, SNP_04,SNP_05 and SNP_06 may all have the resistant donor genotype. Or onlySNP_01 and SNP_02 may have the resistant donor genotype; or only SNP_02and SNP_03 may have the resistant donor genotype; or only SNP_03 andSNP_04 may have the resistant donor genotype, etc. Or only a single SNP,i.e. only SNP_01, or only SNP_02 or only SNP_03, or only SNP_04, or onlySNP_05 or only SNP_06 has the resistant donor genotype. The SNPs that donot have the resistant donor genotype thus have another genotype, therecipient genotype. The recipient genotype for a SNP may be any of theother 3 nucleotides, i.e. for SNP_01 the recipient genotype may beAdenine, Guanine or Thymine. Thus, for example when stating that theintrogression fragment is in-between SNP_03 and SNP_04 regarding the QTLon chromosome 5, SNP_03 and SNP_04 may both have the resistant donorgenotype. Or only a single SNP, i.e. only SNP_03 or only SNP_04 may havethe resistant donor genotype; or even neither SNP_03 nor SNP_04 has theresistant donor genotype, while the sequence in-between still containsQTL5.

The reason that not all of the SNPs provided herein need to have theresistant donor genotype is that the introgression fragment comprisingthe QTL from the donor may be smaller than the chromosome fragmentintrogressed e.g. in the deposited seeds, but the fragment stillcomprises the QTL5. Still, a plant can be recognized to contain theintrogression fragment (comprising the QTL5) by the phenotype, and/or bytransferring the fragment to a susceptible plant and therebytransferring the ToLCNDV resistance phenotype, or by sequencing theregion between the SNP markers to identify the donor fragment, or othermethods known to the skilled person, such as saturating the region withmore SNP markers, allelism tests, identifying the causal gene, etc.

Thus, a combination of methods can be used to show that the QTL5 ispresent in a plant cell or plant, even if not for all of the linked SNPsthe donor SNP genotype is present, QTL5 confers an average ToLCNDVresistance of at least 5.0 when transferred into a susceptible line orvariety and is dominant.

In a preferred embodiment of the invention the introgression fragmentfrom chromosome 5 of the donor plant comprising the sequence of thedonor plant in-between SNP_01 and SNP_06, in-between SNP_01 and SNP_05,in-between SNP_01 and SNP_04, in-between SNP_02 and SNP_06, in-betweenSNP_02 and SNP_05, in-between SNP_02 and SNP_04, in-between SNP_03 andSNP_06, in-between SNP_03 and SNP_05, preferably in-between SNP_03 andSNP_4 confers resistance to ToLCNDV to the cultivated melon plant ceilsaccording to the invention or to the cultivated melon plants accordingto the invention.

Preferably, the melon plant cell according to the invention originatesfrom a cultivated melon plant or the melon plant according to theinvention, is a cultivated melon plant.

In one aspect the present invention relates to cultivated melon plantcells or melon plants (or plant parts) comprising an introgressionfragment from chromosome 5 of a ToLCNDV resistant donor plant, whereinthe introgression fragment confers ToLCNDV resistance and theintrogression fragment is detectable by (comprises) the SNP genotype ofthe donor plant for one or more (or all) of the following SNPs: SNP_01,SNP_02, SNP_03, SNP_04, SNP_05 and or SNP_06, and optionally any SNPin-between SNP_01 and SNP_06.

In one aspect the plant, plant part or plant cell comprises QTL5 andcomprises the SNP donor genotype for at least SNP_03, as this SNP ismost significantly associated with the ToLCNDV resistance QTL5. In afurther aspect the plant, plant part or plant cell comprises the SNPdonor genotype for at least SNP_03 and SNP_04, or for at least SNP_03and SNP_02. Optionally, the plant, plant part or plant cell comprisesQTL5 and comprises the SNP donor genotype for SNP_01, SNP_02 and SNP_03;or for SNP_02, SNP_03 and SNP_04.

An introgression fragment may therefore comprise the donor SNP genotypefor all SNP markers linked to QTL5 (as in the seeds deposited herein),or a smaller fragment, whereby one or more of the SNP markers is notpresent. As described further below, even all or all but one donor SNPmarkers may be absent, while QTL5 is still present on the introgressionfragment.

The nucleotide sequences (SEQ ID NO: 1 to SEQ ID NO: 6) comprising theSNPs provided herein are the nucleotide sequences of the resistantdonor, i.e. they contain the donor SNP nucleotide. Therefore, in oneaspect the present invention relates to cultivated melon plant cells ormelon plants (or plant parts) comprising an introgression fragment fromchromosome 5 of a ToLCNDV resistant donor plant, wherein theintrogression fragment confers ToLCNDV resistance and the introgressionfragment is detectable by (comprises) SEQ ID NO: 1 or an Cytosine atnucleotide 101 of SEQ ID NO: 1 or a Cytosine at the equivalentnucleotide of a sequence having substantial sequence identity to SEQ IDNO: 1 and or by SEQ ID NO: 2 or a Thymine at nucleotide 045 of SEQ IDNO: 2 or a Thymine at the equivalent nucleotide of a sequence havingsubstantial sequence identity to SEQ ID NO:2 and/or by SEQ ID NO: 3 or aThymine at nucleotide 68 of SEQ ID NO: 3 or a Thymine at the equivalentnucleotide of a sequence having substantial sequence identity to SEQ IDNO: 3 and/or by SEQ ID NO: 4 or a Adenine at nucleotide 227 of SEQ IDNO: 4 or a Adenine at the equivalent nucleotide of a sequence havingsubstantial sequence identity to SEQ ID NO: 4 and/or by SEQ ID NO: 5 ora Cytosine at nucleotide 839 of SEQ ID NO; 5 or a Cytosine at theequivalent nucleotide of a sequence having substantial sequence identityto SEQ ID NO; 5 and/or by SEQ ID NO; 6 or a Adenine at nucleotide 445 ofSEQ ID NO: 6 or a Adenine at the equivalent nucleotide of a sequencehaving substantial sequence identity to SEQ ID NO: 6. The ToLCNDVresistance conferring QTL is present on the introgression fragment.

“Donor plant cell” or “donor plant” in connection with the presentinvention shall mean a melon plant cell or melon plant being resistantto ToLCNDV. Likewise, the term DNA fragment or introgression fragmentfrom the donor plant or cell shall mean a fragment of chromosome 5 of amelon plant resistant to ToLCNDV, whereby the fragment confers ToLCNDVresistance when transferred into a TolCNDV susceptible melon plant. In apreferred embodiment of the invention, the donor plant is a wild speciesor wild accession of melon. In a particular preferred embodiment of theinvention, DNA fragments or introgression fragments from donor plantcells or plants are the donor fragments obtained from plants grown fromseeds deposited under NCIMB 42585 or progeny obtained from plants grownfrom seeds deposited under NCIMB 42585 or plants obtained by crosseswith plants grown from seeds deposited under NCIMB 42585.

Donor melon plants can be obtained from various sources. A personskilled in the art knows how to detect other sources of ToLCNDVresistant donor plants. For detecting such sources of ToLCNDV resistantdonor plants, basically melon plants can be infected with ToLCNDV,either by mechanical means, as described in Lopez et al. (2015,Euphytica 204 (3), 679-691) or by transmission of the virus bywhiteflies. Preferably, infection occurs by whitefly infection incontext with the present invention. Plants showing reduced symptomlevels compared to susceptible controls can then be selected and used assource for genome fragments or sequences conferring ToLCNDV resistance.A preferred method on how to infect melon plants with ToLCNDV andmethods for determining the symptom level of infected plants are givenherein under “General Methods”.

In context of the present invention the donor plants preferably have anaverage symptom level equal to or above 5.0, more preferably equal to orabove 6.0, furthermore preferred equal to or above 7.0, even morepreferred equal to or above 8.0 and most preferred equal to or above9.0. In one aspect the donor plant comprises the donor SNP genotype forone or more or all of SNP_01, SNP_2, SNP_3, SNP_04, SNP_05 and SNP_06,as shown in Table 2. Preferably the SNP donor genotype is homozygous.The donor is herein e.g. a wild melon having no agronomic value, e.g.producing white fleshed fruits, having low brix, etc.

“Recurrent plant cell” or “recurrent plant” or “recipient plant” inconnection with the present invention shall be understood to be a melonplant ceil or melon plant being sensitive (used herein synonymously withsusceptible) or non-resistant to ToLCNDV infection. If a plant issensitive or non-resistant to ToLCNDV can be determined by observationof the symptom levels after ToLCNDV infection. A recurrent plantpreferably has an average symptom level below 3.0, more preferably equalto or below 2.5 or equal to or below 2.0. Symptom levels and methods howto infect melon plants with ToLCNDV are described elsewhere herein andare applicable here accordingly. In a preferred embodiment of theinvention, the recurrent melon plant cell according to the inventionoriginates from a cultivated melon plant or the recurrent melon plantaccording to the invention, is a cultivated melon plant. Preferably itis an elite line, breeding line or variety.

“Introgression fragment” refers to a chromosome fragment, chromosomepart or region which has been introduced into another plant of the sameor related species by crossing or traditional breeding techniques. Theintrogression of the fragment from a donor plant into a recurrent plantintroduces into the offspring of a cross between the donor and recurrentplant a phenotype, which was not present in the recurrent plant.Concerning the present invention, the phenotype transferred from thedonor plant to the recurrent plant is resistance to ToLCNDV, e.g. anaverage disease score of 5.0. For introgression of a fragment into aspecific breeding line or variety the first crossing step can e.g. befollowed by one or more back crossings with the intended breeding lineor variety. As understood herein, introgression can mean a firstcrossing of a ToLCNDV resistant donor plant with a ToLCNDV non-resistantrecurrent plant and further back-crossing one or several times ToLCNDVresistant plants obtained from the first crossing with plants of therecipient into which ToLCNDV resistance shall be introgressed. In such acase, the introgressed fragment is the result of breeding methodsreferred to by the verb “to introgress” (such as backcrossing) into arecipient variety or breeding line. Thus, introgression of ToLCNDVresistance into a recurrent plant is a technical process directed byman. In particular introgression herein refers to a manmade breedingprocess or method. One or more or all of the molecular markers (SNPmarkers) provided herein can be used in that process. The resultingplant, i.e. the cultivated line or variety comprising one introgressionfragment (on chromosome 5) from a donor, i.e. comprising a recombinantchromosome 5, is also man-made and does not exist in nature.

The introgression fragment can be large, e.g. even half of a chromosome,but is preferably smaller, such as about 15 Mb or less, such as about 10Mb or less, about 9 Mb or less, about 8 Mb or less, about 7 Mb or less,about 6 Mb or less, about 5 Mb or less, about 4 Mb or less, about 3 Mbor less, about 2 Mb or less, about 1 Mb (equals 1,000,000 base or less),or about 0.8 Mb (equals 800,000 base pairs) or less.

The introgression fragment can originate from a wild melon plant or wildmelon accession or wild relatives of melon or landraces (donor). Wildmelon plants or wild melon accessions or wild relatives of melon plantsor landraces can be used to introgress fragments of the donor genomeinto the genome of cultivated melon, Cucumis melo, to generate breedinglines or varieties with good agronomic characteristics. Such acultivated melon plant thus has a “genome of cultivated C. melo”, butcomprises in its genome a fragment of a donor, e.g. an introgressionfragment of a related wild Cucumis genome, such as Cucumis melo ssp.agrestis, C. melo ssp. melo, C. melo ssp. acidulous, C. callosus, C.trigonus, C. picrocarpus, or another wild melon or wild relative ofmelon. It is understood that the term “introgression fragment” neverincludes a whole chromosome, but only a part of a chromosome. Thechromosomes carrying the introgression therefore also comprise a part orparts of the recurrent (recipient) melon plant and in addition parts ofthe donor melon plant.

When the chromosome 5 of cultivated melon comprises an introgressionfragment, this therefore means that the cultivated melon plant comprisesa recombinant chromosome 5, whereby the introgressed fragment comprisesthe ToLCNDV resistance conferring QTL. As described elsewhere, theintrogression fragment from the donor may comprise one or more or all ofthe donor SNP nucleotides (for SNP_01, SNP_02, SNP_03, SNP_04, SNP_05and/or SNP_06) or one or more or all of the sequences comprising thedonor SNP nucleotides (SEQ ID NO: 1, 2, 3, 4, 5 and or SEQ ID NO: 6).

So, e.g. the introgression fragment (in homozygous or heterozygous form)may comprise, and is detectable by, one or more or all of the followingSNP genotypes: the CC or CT genotype for SNP_01 in SEQ ID NO: 1, and orthe TT or TG genotype for SNP_02 in SEQ ID NO: 2, and, or the TT or TCgenotype for the SNP_03 in SEQ ID NO: 3 and/or the AA or AG genotype forthe SNP_04 in SEQ ID NO: 4, and, or the CC or CT genotype for SNP_05 inSEQ ID NO: 5, and, or the AA or AG genotype for SNP_06 in SEQ ID NO: 6.

In the instant invention, in one aspect the lower part of chromosome 5of cultivated melon, below marker CMGAAN144 on chromosome 5 on page 3 ofDiaz et al 2015 (Mol. Breeding 35: 188), or between marker CMGAAN144 andmarker A1__13-H12, comprises QTL5 from a donor.

In one aspect the donor plant of the invention is not one of the fiveCucumis melo subsp. agrestis accessions (subsp. agrestis var. momordica:Mom-Khalnd/Kharbuja, Mom-PI124Ind/PI124112, Mom-PI124Ind/PI414723 andsubsp. agrestis wild types: Ag-WM9Ind/WM9, Ag-WM7Ind/WM7) resistant toToLCNDV identified by Lopez et al., 2015, Euphytica 204 (3), 679-691.

The term “breeding” encompasses herein crossing, backcrossing, selfing,

selection, double haploid production, embryo rescue, protoplast fusion,marker assisted selection, mutation breeding etc. as known to thebreeder (i.e. methods other than geneticmodification/transformation/transgenic methods), by which, for example,a recombinant chromosome 5 can be obtained, identified, produced and/ortransferred.

In a preferred embodiment of the present invention, the introgressionfragment originates form a wild Cucumis plant or a wild Cucumisaccession, most preferably the introgression fragment originates fromwild Cucumis melo ssp. melo, having small fruits (no more than 6 cmlong), with white, acid tasting, fruit flesh. This donor was used in theinstant invention, but other donors can be identified by the skilledperson which comprise e.g. the same SNP genotype as this donor forSNP_01 to SNP_06 and comprises a QTL in the same region of chromosome 6.

A suitable donor is in one aspect a wild C. melo plant or accessionhaving an average ToLCNDV disease score of at least 7.0, or at least7.1, 7.2, 7.3, or 7.4 or 7.5 or 7.6, on a scale of 1—dead plant to 9—nosymptoms and comprises one or more or all of the following SNPgenotypes:

the CC or CT genotype for SNP_01 in SEQ ID NO: 1, and/or the TT or TGgenotype for SNP_02 in SEQ ID NO: 2, and/or the TT or TC genotype forthe SNP_03 in SEQ ID NO: 3 and/or the AA or AG genotype for the SNP_04in SEQ ID NO: 4, and/or the CC or CT genotype for SNP_05 in SEQ ID NO:5, and or the AA or AG genotype for SNP_06 in SEQ ID NO: 6.

“Plant variety” is a group of plants within the same botanical taxon ofthe lowest grade known, which (irrespective of whether the conditionsfor the recognition of plant breeder's rights are fulfilled or not) canbe defined on the basis of the expression of characteristics that resultfrom a certain genotype or a combination of genotypes, can bedistinguished from any other group of plants by the expression of atleast one of those characteristics, and can be regarded as an entity,because it can be multiplied without any change. Therefore, the term“plant variety” cannot be used to denote a group of plants, even if theyare of the same kind, if they are all characterized by the presence ofone or two loci or genes (or phenotypic characteristics due to thesespecific loci or genes), but which can otherwise differ from one anotherenormously as regards the other loci or genes.

“F1, F2, F3, etc.” refers to the consecutive related generationsfollowing a cross between two parent plants or parent lines. The plantsgrown from the seeds produced by crossing two plants or lines is calledthe F1 generation. Selfing the F1 plants results in the F2 generation,etc.

“F1 hybrid” plant (or F1 hybrid seed) is the generation obtained fromcrossing two inbred parent lines. Thus, F1 hybrid seeds are seeds fromwhich F1 hybrid plants grow, F1 hybrids are more vigorous and higheryielding, due to heterosis. Inbred lines are essentially homozygous atmost loci in the genome.

A “plant line” or “breeding line” refers to a plant and its progeny. Asused herein, the term “inbred line” refers to a plant line which hasbeen repeatedly selfed and is nearly homozygous. Thus, an “inbred line”or “parent line” refers to a plant which has undergone severalgenerations (e.g. at least 5, 6, 7 or more) of inbreeding, resulting ina plant line with a high uniformity.

“Uniformity” or “uniform” relates to the genetic and phenotypiccharacteristics of a plant line or variety. Inbred lines are geneticallyhighly uniform as they are produced by several generations ofinbreeding. Likewise, and the F1 hybrids which are produced from suchinbred lines are highly uniform in their genotypic and phenotypiccharacteristics and performance.

In a particular preferred embodiment of the invention, plant cellsaccording to the invention and plants according to the invention arecharacterized in that the introgression fragment conferring ToLCNDVresistance originates from the seeds deposited under NCIMB 42585 orprogeny thereof.

In a further preferred embodiment of the invention, the melon plant cellaccording to the invention originates from a cultivated melon plant orthe melon plant according to the invention is a cultivated melon plantand the introgression fragment originates form a wild Cucumis plant or awild Cucumis accession or from donor plants described herein to bepreferred donor plants or obtained from seeds deposited under NCIMB42585 or progeny thereof.

“Chromosome 5 of a melon plant” is to be understood in context of thepresent invention as the scaffolds, fragments, regions, markers andnucleic acid sequences assigned by the ICuGI (International CucurbitGenomics Initiative) to belong to chromosome 5 of the melon genome.

“Orthologous chromosome 5” refers to the chromosome 5 of wild relativesof melon, parts of which can be introgressed into cultivated melonchromosome 5.

A “recombinant chromosome” refers to a chromosome having a new geneticmakeup arising through crossing over between homologous chromosomes,e.g. a “recombinant chromosome 5”, i.e. a chromosome 5 which is notpresent in either of the parent plants and arose through a rarecrossing-over event between homologous chromosomes of a chromosome 5pair. Herein, for example, a recombinant melon chromosome 5 comprising aToLCNDV-resistance conferring QTL is provided. The recombinantchromosome 5 therefore is a chromosome of cultivated melon, with anintrogression fragment from a wild donor, whereby the introgressionfragment comprises the ToLCNDV resistance conferring QTL.

“ICuGI” refers herein to the Cucumis melo data published by theInternational Cucurbit Genomics Initiative, which publishes genetic mapsof e.g. Cucumis melo (world wide web at icugi.org/cgi-bin/cmap/mapset_info?species acc=CM). The current version CM_3.5.1 of the C. melogenome map is of Mar. 4, 2012 and the map of chromosome 5 is referred toas ICuGI_V (or LG_V, or Linkage Group V). Further information includingadditional markers and mapping information in addition to the ICuCI datais available from Diaz et al. (2015, Mol Breeding 35, 188) and theadditional data included in the online version of the respectivearticle.

“Cultivated melon plant” refers to plants of Cucumis melo i.e.varieties, breeding lines or cultivars of the species C. melo,cultivated by humans and having good agronomic characteristics,especially producing edible and marketable fruits of good size andquality and uniformity; such plants are not “wild melon plants”, i.e.plants which generally have much poorer yields and poorer agronomiccharacteristics than cultivated plants and e.g. grow naturally in wildpopulations. “Wild plants” include for example ecotypes, PI (PlantIntroduction) lines, landraces or wild accessions or wild relatives of aspecies.

A “SNP (=Single Nucleotide Polymorphism)” in context with the presentinvention is to be understood as a variation in a single nucleotide thatoccurs at a specific position in the genome. A SNP is the variation ofthe single nucleotide at the given position in a genome between twoplants, if a wild melon plant having a ToLCNDV resistance (donor plant)shows in its corresponding sequence at a specific single position anucleotide which is different from the corresponding nucleotide at thesame position of a cultivated melon plant, the position defines a SNPbetween the wild melon and the cultivated melon. If the donor plant hasone of the four possible nucleotides (A, C, T or G) at a specificposition, a SNP occurs, when the cultivated plant has either of theremaining three possible nucleotides at the same corresponding sequenceposition. In a cultivated melon plant comprising an introgressionfragment from a donor, it can therefore easily be determined if thesingle nucleotide of the SNP is from the donor or from the cultivatedmelon (recipient).

‘SNP nucleotide’ refers to the single nucleotide, while ‘SNP genotype’refers to the pair of nucleotides in a diploid plant cell. So, forSNP_01, the SNP nucleotide of the ToLCNDV resistant donor is a Cytosine(C) for nucleotide 501 of SEQ ID NO: 1, while the SNP genotype of aplant or cell comprising SEQ ID NO: 1 can be CC (Cytosine on bothchromosomes) or CP (Cytosine on one chromosome and Thymine on the otherchromosome), whereby the ToLCNDV resistant donor SNP nucleotide(Cytosine), and thus SEQ ID NO: 1 (or a sequence substantially identicalto SEQ ID NO: 1), is homozygous or heterozygous. The term “SNP donorgenotype” refers to the donor SNP nucleotide being present in homozygousor in heterozygous form, i.e. for SNP_01 the SNP donor genotype iseither CC or CT.

“SNP_01” which is alternatively designated “mME11320_k” is to beunderstood in context with the present invention to be a SNP at position101 in SEQ ID NO 1, SEQ ID NO 5 or a sequence substantially identical toSEQ ID NO 1 can be found on chromosome 5 in the ICuGI data set. Therelative position of SNP_05 according to markers published by ICuGI isderivable from Table 5. Preferably the nucleotide sequence comprisingSNP_05 has a nucleotide sequence having at least 85% identity with thenucleotide sequence shown under SEQ ID NO 1, more preferably at least90% identity with the nucleotide sequence shown under SEQ ID NO 1,further more preferably at least 95% identity with the nucleotidesequence shown under SEQ ID NO 1, even more preferred at least 97%identity with the nucleotide sequence shown under SEQ ID NO 1, evenfurther more preferred at least 98% identity with the nucleotidesequence shown under SEQ ID NO 1, in particular preferred at least 99%identity with the nucleotide sequence shown under SEQ ID NO 1 or moreparticularly preferred at least 99.5% identity with the nucleotidesequence shown under SEQ ID NO 1 under the provision that in each casethe nucleotide at position 101 in SEQ ID NO 1 is different from thecorresponding nucleotide at the same position of the recurrent plant.Such sequences having at least 85%, 90%, 95%, 97%, 98%, 99% or moresequence identity to SEQ ID NO: 1 are referred to as having substantialsequence identity to SEQ ID NO: 1.

The ToLCNDV resistant donor plants used in the invention have a ‘C’(Cytosine) at position 101 in SEQ ID NO 1 or in a sequence comprisingsubstantial sequence identity to SEQ ID NO:1. In a preferred embodimentof the invention, SNP_01 is characterized in that the recurrent planthas an A, G or T at position 101 in SEQ ID NO 1. In one aspect, therecurrent plant has a T at position 101 in SEQ ID NO 1.

In one embodiment of the invention, SNP_01 is characterized in that thedonor plant has a C at position 101 of SEQ ID NO 1 (or at the equivalentposition of a sequence comprising substantial sequence identity to SEQID NO: 1) and the recurrent plant has a T at position 101 in SEQ ID NO1.

“SNP_02” which is alternatively designated “mME43070_k” is to beunderstood in context with the present invention to be a SNP at position945 in SEQ ID NO 2, SEQ ID NO 2 or a sequence substantially identical toSEQ ID NO 2 can be found on chromosome 3 in the ICuGI data set. Therelative position of SNP_02 according to markers published by ICuGI isderivable from Table 1, Preferably the nucleotide sequence comprisingSNP_02 has a nucleotide sequence having at least 85% identity with thenucleotide sequence shown under SEQ ID NO 2, more preferably at least90% identity with the nucleotide sequence shown under SEQ ID NO 2,further more preferably at least 95% identity with the nucleotidesequence shown under SEQ ID NO 2, even more preferred at least 97%identity with the nucleotide sequence shown under SEQ ID NO 2, evenfurther more preferred at least 98% identity with the nucleotidesequence shown under SEQ ID NO 2, in particular preferred at least 99%identity with the nucleotide sequence shown under SEQ ID NO 2 or moreparticularly preferred at least 99.5% identity with the nucleotidesequence shown under SEQ ID NO 2 under the provision that in each casethe nucleotide at position 945 in SEQ ID NO 2 is different from thecorresponding nucleotide at the same position of the recurrent plant.Such sequences having at least 85%, 90%, 95%, 97%, 98%, 99% or moresequence identity to SEQ ID NO 2 are referred to as having substantialsequence identity to SEQ ID NO: 2.

The ToLCNDV resistant donor plants used in the invention have a ‘T’(Thymine) at position 945 in SEQ ID NO 2 or in a sequence comprisingsubstantial sequence identity to SEQ ID NO: 2.

In one embodiment of the invention, SNP_02 is characterized in that therecurrent plant has an A, C or G at position 945 in SEQ ID NO 2 or in asequence comprising substantial sequence identity to SEQ ID NO: 2.

In one aspect the recurrent plant has a ‘G’ (Guanine) at position 945 inSEQ ID NO 2 or in a sequence comprising substantial sequence identity toSEQ ID NO: 2.

In a particular embodiment of the invention, SNP_02 is characterized inthat the donor plant has a T at position 945 in SEQ ID NO 2 (or in asequence comprising substantial sequence identity to SEQ ID NO: 2) andthe recurrent plant has a G at position 945 in SEQ ID NO 2 (or in asequence comprising substantial sequence identity to SEQ ID NO: 2).

“SNP_03” which is alternatively designated “mME10621_k” is to beunderstood in context with the present invention to be a SNP at position68 in SEQ ID NO 3, SEQ ID NO 3 or a sequence substantially identical toSEQ ID NO 3 can be found on chromosome 5 in the ICuGI data set. Therelative position of SNP_03 according to markers published by ICuGI isderivable from Table 1. Preferably the nucleotide sequence comprisingSNP_03 has a nucleotide sequence having at least 85% identity with thenucleotide sequence shown under SEQ ID NO 3, more preferably at least90% identity with the nucleotide sequence shown under SEQ ID NO 3,further more preferably at least 95% identity with the nucleotidesequence shown under SEQ ID NO 3, even more preferred at least 97%identity with the nucleotide sequence shown under SEQ ID NO 3, evenfurther more preferred at least 98% identity with the nucleotidesequence shown under SEQ ID NO 3, in particular preferred at least 99%identity with the nucleotide sequence shown under SEQ ID NO 3 or moreparticularly preferred at least 99.5% identity with the nucleotidesequence shown under SEQ ID NO 3 under the provision that in each casethe nucleotide at position 68 in SEQ ID NO 3 is different from thecorresponding nucleotide at the same position of the recurrent plant.Such sequences having at least 85%, 90%, 95%, 97%, 98%, 99% or moresequence identity to SEQ ID NO 3 are referred to as having substantialsequence identity to SEQ ID NO: 3.

The ToLCNDV resistant donor plants used in the invention have a ‘T’(Thymine) at position 68 in SEQ ID NO 3 (or in a sequence comprisingsubstantial sequence identity to SEQ ID NO: 3). In one embodiment of theinvention, SNP_03 is characterized in that the recurrent plant has an A,C or G at position 68 in SEQ ID NO 3 (or in a sequence comprisingsubstantial sequence identity to SEQ ID NO: 3). In one aspect therecurrent plant has a C position 68 in SEQ ID NO 3 (or in a sequencecomprising substantial sequence identify to SEQ ID NO: 3).

In one embodiment of the invention, SNP_03 is characterized in that thedonor plant has a ‘T’ at position 68 in SEQ ID NO 3 (or in a sequencecomprising substantial sequence identity to SEQ ID NO: 3) and therecurrent plant has a ‘C’ at position 68 in SEQ ID NO 3 (or in asequence comprising substantial sequence identity to SEQ ID NO: 3).

“SNF_04” which is alternatively designated “mME50729_k” is to beunderstood in context with the present invention to be a SNP at position227 in SEQ ID NO 4, SEQ ID NO 4 or a sequence substantially identical toSEQ ID NO 4 can be found on chromosome 5 in the ICuGI data set. Therelative position of SNP_04 according to markers published by ICuGI isderivable from Table 1. Preferably the nucleotide sequence comprisingSNP_04 has a nucleotide sequence having at least 85% identity with thenucleotide sequence shown under SEQ ID NO 4, more preferably at least90% identity with the nucleotide sequence shown under SEQ ID NO 4,further more preferably at least 95% identity with the nucleotidesequence shown under SEQ ID NO 4, even more preferred at least 97%identity with the nucleotide sequence shown under SEQ ID NO 4, evenfurther more preferred at least 98% identity with the nucleotidesequence shown under SEQ ID NO 4, in particular preferred at least 99%identity with the nucleotide sequence shown under SEQ ID NO 4 or moreparticularly preferred at least 99.5% identity with the nucleotidesequence shown under SEQ ID NO 4 under the provision that in each casethe nucleotide at position 227 in SEQ ID NO 4 is different from thecorresponding nucleotide at the same position of the recurrent plant.Such sequences having at least 85%, 90%, 95%, 97%, 98%, 99% or moresequence identity to SEQ ID NO 4 are referred to as having substantialsequence identity to SEQ ID NO: 4.

The ToLCNDV resistant donor plants used in the invention have an ‘A’(Adenine) at position 227 in SEQ ID NO 4 (or in a sequence comprisingsubstantial sequence identity to SEQ ID NO: 4). In one embodiment of theinvention, SNP_04 is characterized in that the recurrent: plant has anC, T or G at position 227 in SEQ ID NO 4 (or in a sequence comprisingsubstantial sequence identity to SEQ ID NO: 4). In one aspect therecurrent plant has a G position 227 in SEQ ID NO 4 (or in a sequencecomprising substantial sequence identity to SEQ ID NO: 4).

In one embodiment of the invention, SNP_04 is characterized in that theToLCNDV resistant donor plant has an A at position 227 in SEQ ID NO 4(or in a sequence comprising substantial sequence identity to SEQ ID NO:4) and the recurrent plant has a G at position 227 in SEQ ID NO 4 (or ina sequence comprising substantial sequence identity to SEQ ID NO: 4).

“SNP_05” which is alternatively designated “mME32395_k” is to beunderstood in context with the present invention to be a SNP at position839 in SEQ ID NO 5, SEQ ID NO 5 or a sequence substantially identical toSEQ ID NO 5 can be found on chromosome 5 in the ICuGI data set. Therelative position of SNP_05 according to markers published by ICuGI isderivable from Table 1. Preferably the nucleotide sequence comprisingSNP_05 has a nucleotide sequence having at least 85% identity with thenucleotide sequence shown under SEQ ID NO 5, more preferably at least90% identity with the nucleotide sequence shown under SEQ ID NO 5,further more preferably at least 95% identity with the nucleotidesequence shown under SEQ ID NO 5, even more preferred at least 97%identity with the nucleotide sequence shown under SEQ ID NO 5, evenfurther more preferred at least 98% identity with the nucleotidesequence shown under SEQ ID NO 5, in particular preferred at least 99%identity with the nucleotide sequence shown under SEQ ID NO 5 or moreparticularly preferred at least 99.5% identity with the nucleotidesequence shown under SEQ ID NO 5 under the provision that in each casethe nucleotide at position 839 in SEQ ID NO 5 is different from thecorresponding nucleotide at the same position of the recurrent plant.Such sequences having at least 85%, 90%, 95%, 97%, 98%, 99% or moresequence identity to SEQ ID NO 5 are referred to as having substantialsequence identity to SEQ ID NO: 5.

The ToLCNDV resistant donor plants used in the invention have a ‘C’(Cytosine) at position 839 in SEQ ID NO 5 (or in a sequence comprisingsubstantial sequence identity to SEQ ID NO: 5). In one embodiment of theinvention, SNP_05 is characterized in that the recurrent plant has an A,T or G at position 839 in SEQ ID NO 5 (or in a sequence comprisingsubstantial sequence identity to SEQ ID NO: 5). In one aspect therecurrent plant has a T position 839 in SEQ ID NO 5 (or in a sequencecomprising substantial sequence identity to SEQ ID NO: 5).

In one embodiment of the invention, SNP_05 is characterized in that theToLCNDV resistant donor plant has a C at position 839 in SEQ ID NO 5 (orin a sequence comprising substantial sequence identity to SEQ ID NO: 5)and the recurrent plant has a T at position 839 in SEQ ID NO 5 (or in asequence comprising substantial sequence identity to SEQ ID NO: 5).

“SNP_06” which is alternatively designated “mME49184_k” is to beunderstood in context with the present invention to be a SNP at position443 in SEQ ID NO 6. SEQ ID NO 6 or a sequence substantially identical toSEQ ID NO 6 can be found on chromosome 5 in the ICuGI data set. Therelative position of SNP_06 according to markers published by ICuGI isderivable from Table 1. Preferably the nucleotide sequence comprisingSNP_06 has a nucleotide sequence having at least 85% identity with thenucleotide sequence shown under SEQ ID NO 6, more preferably at least90% identity with the nucleotide sequence shown under SEQ ID NO 6,further more preferably at least 95% identity with the nucleotidesequence shown under SEQ ID NO 6, even more preferred at least 97%identity with the nucleotide sequence shown under SEQ ID NO 6, evenfurther more preferred at least 98% identity with the nucleotidesequence shown under SEQ ID NO 6, in particular preferred at least 99%identity with the nucleotide sequence shown under SEQ ID NO 6 or moreparticularly preferred at least 99.5% identity with the nucleotidesequence shown under SEQ ID NO 6 under the provision that in each casethe nucleotide at position 445 in SEQ ID NO 6 is different from thecorresponding nucleotide at the same position of the recurrent plant.Such sequences having at least 85%, 90%, 95%, 97%, 98%, 99% or moresequence identity to SEQ ID NO 6 are referred to as having substantialsequence identity to SEQ ID NO: 6.

The ToLCNDV resistant donor plants used in the invention have an ‘A’(Adenine) at position 445 in SEQ ID NO 6 (or in a sequence comprisingsubstantial sequence identity to SEQ ID NO: 6). In one embodiment of theinvention, SNP_06 is characterized in that the recurrent plant has a C,T or G at position 445 in SEQ ID NO 6 (or in a sequence comprisingsubstantial sequence identity to SEQ ID NO: 6). In one aspect therecurrent plant has a G position 445 in SEQ ID NO 6 (or in a sequencecomprising substantial sequence identity to SEQ ID NO: 6).

In a particular embodiment of the invention, SNP_06 is characterized inthat the ToLCNDV resistant donor plant has an A at position 445 in SEQID NO 6 (or in a sequence comprising substantial sequence identity toSEQ ID NO: 6) and the recurrent plant has a G at position 445 in SEQ IDNO 6 (or in a sequence comprising substantial sequence identity to SEQID NO: 6).

TABLE 1 Pseudo- cM Garcia- Scaffold v3.5.1 Marker molecule Mas et al. cMArgyris et (world wide web at Scaffold Marker name type Chromcoordinates (2012) al. (2015) LG_ICuGI cM_ICuGI melonomics.net)coordinates CMPSNP460 SNP 5 12.966.671 57.3 CM3.5.1_scaffold000091.816.109 SNP_06 SNP 5 CM3.5.1_scaffold00009 2.607.437 CMPSNP682 SNP 515.854.444 57.3 50.4 CM3.5.1_scaffold00009 4.703.882 CMN62_05 SSR 516.570.623 CM3.5.1_scaffold00009 5.420.061 CMPSNP1005 SNP 5 17.239.62157.3 50.4 CM3.5.1_scaffold00009 6.089.059 CMN61_15 SSR 5 17.453.772CM3.5.1_scaffold00009 6.303.210 CMPSNP39 SNP 5 17.651.074 57.3CM3.5.1_scaffold00009 6.500.512 PS_03-B08 SNP 5 18.119.560 57.3 50.4CM3.5.1_scaffold00009 6.968.998 ECM203 SSR 5 18.459.732CM3.5.1_scaffold00009 7.309.170 CMPSNP2005 SNP 5 18.833.037 50.4CM3.5.1_scaffold00009 7.682.475 CMN01_49 SSR 5 19.305.580CM3.5.1_scaffold00003 26.051 ECM142 SSR 5 19.743.282 5 54CM3.5.1_scaffold00003 463.753 CMGAN3 SSR 5 19.871.767 5 56CM3.5.1_scaffold00003 592.238 CMPSNP1018 SNP 5 20.088.198 50.9 50.7CM3.5.1_scaffold00003 808.669 CMPSNP613 SNP 5 20.406.864CM3.5.1_scaffold00003 1.127.335 CMN05_89 SSR 5 21.514.552CM3.5.1_scaffold00003 2.235.023 CMN23_06 SSR 5 21.949.502CM3.5.1_scaffold00003 2.669.973 GCM295 SSR 5 23.318.454CM3.5.1_scaffold00003 4.038.925 CMPSNP741 SNP 5 23.620.132CM3.5.1_scaffold00003 4.340.603 ECM115 SSR 5 23.786.719CM3.5.1_scaffold00003 4.507.190 CMPSNP1136 SNP 5 24.100.004 67.0 67.7CM3.5.1_scaffold00003 4.820.475 ECM206 SSR 5 24.175.232CM3.5.1_scaffold00003 4.895.703 CMCTN2 SSR 5 24.313.118 5 73CM3.5.1_scaffold00003 5.033.589 CMGAAN144 SSR 5 24.403.405 5 73CM3.5.1_scaffold00003 5.123.876 SNP_05 SNP 5 CM3.5.1_scaffold000035.202.092 3J84-496 SNP 5 24.884.331 CM3.5.1_scaffold00003 5.604.8023J84-19 SNP 5 24.884.834 CM3.5.1_scaffold00003 5.605.305 60k53-404 SNP 525.036.462 CM3.5.1_scaffold00003 5.756.933 60k53-147 SNP 5 25.036.692CM3.5.1_scaffold00003 5.757.163 60k53-80 SNP 5 25.036.780CM3.5.1_scaffold00003 5.757.251 60k49-351 SNP 5 25.040.592CM3.5.1_scaffold00003 5.761.063 60k49-307 SNP 5 25.040.689CM3.5.1_scaffold00003 5.761.160 60k49-182 SNP 5 25.040.770CM3.5.1_scaffold00003 5.761.241 60k45.389 SNP 5 25.043.452 73.4CM3.5.1_scaffold00003 5.763.923 60k45.288 SNP 5 25.043.521CM3.5.1_scaffold00003 5.763.992 60k45.213 SNP 5 25.043.624CM3.5.1_scaffold00003 5.764.095 60k45.14 SNP 5 25.043.807 73.4CM3.5.1_scaffold00003 5.764.278 60k42-490 SNP 5 25.045.798CM3.5.1_scaffold00003 5.766.269 60k42-411 SNP 5 25.045.907CM3.5.1_scaffold00003 5.766.378 60k42-126 SNP 5 25.046.183 76.6CM3.5.1_scaffold00003 5.766.654 60k42-29 SNP 5 25.046.269CM3.5.1_scaffold00003 5.766.740 60k41.333 SNP 5 25.047.374CM3.5.1_scaffold00003 5.767.845 60k41.243 SNP 5 25.047.449 73.4CM3.5.1_scaffold00003 5.767.920 60k41.49 SNP 5 25.047.654CM3.5.1_scaffold00003 5.768.125 SNP_04 SNP 5 CM3.5.1_scaffold000035.785.550 CMTAAN128 SSR 5 25.081.134 5 72 CM3.5.1_scaffold000035.801.605 CMTAN138 SSR 5 25.081.134 5 70 CM3.5.1_scaffold00003 5.801.605CMTAN139 SSR 5 25.081.134 5 70 CM3.5.1_scaffold00003 5.801.605 CMPSNP588SNP 5 25.771.831 75.0 78.3 CM3.5.1_scaffold00003 5.992.302 CMPSNP464 SNP5 25.639.164 75.8 CM3.5.1_scaffold00003 6.359.635 SNP_03 SNP 5CM3.5.1_scaffold00003 6.553.350 CMPSNP1155 SNP 5 26.124.693 79.8CM3.5.1_scaffold00003 6.845.164 SNP_02 SNP 5 CM3.5.1_scaffold000036.950.286 CMPSNP690 SNP 5 26.340.629 79.8 90.0 CM3.5.1_scaffold000037.061.100 CMTCN227 SSR 5 26.453.003 5 61 CM3.5.1_scaffold00003 7.173.474CMPSNP1115 SNP 5 26.639.938 84.6 CM3.5.1_scaffold00003 7.360.409 ECM213SSR 5 26.907.003 CM3.5.1_scaffold00003 7.627.474 SNP_01 SNP 5CM3.5.1_scaffold00003 7.824.960 AI_13-H12 SNP 5 27.276.249 89.4CM3.5.1_scaffold00003 7.996.720

The molecular markers described herein may be detected according tostandard methods. For example SNP markers can be detected using aKASP-assay (see world wide web at kpbioscience.co.uk) or other assays. AKASP-assay has been developed for SNPs described herein. Respectivedetails are disclosed in the Example section. Sequences used in therespective KASP-assays are given in the Sequence Listing. For developingKASP-assays for the SNPs two allele specific forward primers and oneallele specific reverse primer were designed according to common generalknowledge (see e.g. Allen et al. 2011, Plant Biotechnology J. 9, 1 086-1 099, especially p097-098 for KASP assay method).

“Sequence identity” and “sequence similarity” can be determined byalignment of two peptide or two nucleotide sequences using global orlocal alignment algorithms. Sequences may then be referred to as“substantially identical” or “essentially similar” when they areoptimally aligned by for example the programs GAP or BESTFIT or theEmboss program “Needle” (using default parameters, see below) share atleast a certain minimal percentage of sequence identity defined furtherbelow). These programs use the Needleman and Wunsch global alignmentalgorithm to align two sequences over their entire length, maximizingthe number of matches and minimizes the number of gaps. Generally, thedefault parameters are used, with a gap creation penalty=10 and gapextension penalty=0.5 (both for nucleotide and protein alignments). Fornucleotides the default scoring matrix used is DNAFULL and for proteinsthe default scoring matrix is Blosum62 (Henikoff & Henikoff, 1 992, PNAS89, 1 09 1 5-1 09 1 9). Sequence alignments and scores for percentagesequence identity may for example be determined using computer programs,such as EMBOSS as available on the world wide web underebi.ac.uk:Tools/psa/emboss_needle). Alternatively sequence similarity oridentity may be determined by searching against databases such as FASTA,BLAST, etc., but bits should be retrieved and aligned pairwise tocompare sequence identity. Two proteins or two protein domains, or twonucleic acid sequences have “substantial sequence identity” if thepercentage sequence identity is at least 85%, 90%, 95%, 98%, 99% or more(e.g. at least 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 ormore (as determined by Emboss “needle” using default parameters, i.e.gap creation penalty=10, gap extension penalty=0.5, using scoring matrixDNAFULL for nucleic acids an Blosum62 for proteins).

When reference is made to a nucleic acid sequence (e.g. DMA or genomicDNA) having “substantial sequence identity to” a reference sequence orhaving a sequence identity of at least 80%, e.g. at least 85%, 90%, 95%,98%, 99%, 99.2%, 99.5%, 99.9% nucleic acid sequence identify to areference sequence, in one embodiment said nucleotide sequence isconsidered substantially identical to the given nucleotide sequence andcan be identified using stringent hybridization conditions. In anotherembodiment, the nucleic acid sequence comprises one or more mutationscompared to the given nucleotide sequence but still can be identifiedusing stringent hybridization conditions.

Cultivated melon plants comprising a ToLCNDV resistance conferringfragment introgressed on chromosome 5 show reduced symptoms wheninfected with ToLCNDV, while susceptible controls (lacking theintrogression fragment) show the expected severe symptoms in the sameconditions.

In a preferred embodiment of the invention, plant cells according to theinvention or plants according to the invention are characterized in thatthey upon infection with ToLCNDV show an average symptom level of atleast 4.0, more preferably at least 5.0. As mentioned previously, QTL5confers resistance when the QTL is in homozygous or heterozygous form. AToLCNDV susceptible plant into which the introgression fragmentcomprising QTL5 is introduced by e.g. backcrossing and which isoptionally selfed to generate a homozygous introgression fragment(comprising the donor genotype for one or more or all of SNP_01, SNP_02,SNP_03, SNP_04, SNP_05 and SNP_06 in homozygous form), will result in aplant which is resistant to ToLCNDV infection, having an average diseasescore of at least 4.0, preferably at least 5.0, preferably at least 6.0or preferably at least 7.0. Symptom levels occurring; after infectionwith ToLCNDV between 1 and 9, wherein 1 is defined to be the level withthe most severe symptoms and 9 is defined as the highest resistancelevel, have been described herein above and are applicable hereaccordingly. A preferred test for determining the symptom levels isgiven below under “General Methods”.

In another embodiment of the invention plant cells according to theinvention or plants according to the invention are characterized in thatthey upon infection with ToLCNDV show a symptom level between 4 and 6,more preferably a symptom level of between 5 and 6, while thesusceptible controls show a symptom level of 2 or less.

Resistance to ToLCNDV conferred by the introgression fragment isexpressed in a dominant manner and thus can be observed when only onechromosome 5 comprises the introgression fragment comprising thesequence of the donor plant between the markers of chromosome 5disclosed herein.

Other specific embodiments of the present invention therefore relate tomelon plant cells according to the invention, or melon plants accordingto the invention, wherein the introgression fragment from chromosome 5of a ToLCNDV resistant donor plant comprising the sequence of the donorplant in-between SNP_01 and SNP_06, in-between SNP_01 and SNP_05,in-between SNP_01 and SNP_04, in-between SNP_02 and SNP_06, in-betweenSNP_02 and SNP_05, in-between SNP_02 and SNP_04, in-between SNP_03 andSNP_06, in-between SNP_03 and SNP_05 or preferably in-between SNP_03 andSNP_04 is present in heterozygous state. Accordingly, it is sufficientthat at least one chromosome 5 in the plant cells according to theinvention or plants according to the invention comprise the justdescribed introgression fragment. It is however understood, thatchromosome 5 in respect to the just described introgression fragment canalso be present in the homozygous state without diminishing the degreeof resistance, because of the dominance of the ToLCNDV resistanceconferred by the just described introgression fragment. Thus, theinvention comprises plant cells according to the invention or plantsaccording to the invention which comprise the just describedintrogression fragment in a heterozygous or homozygous state.

Table 2 illustrates the SNP genotype of plants or cells comprising theToLCNDV resistant donor SNPs in homozygous form of heterozygous form, aswell as the recurrent parent SNP genotype, lacking the introgressionfragment.

SNP geno- SNP geno- SNP geno- type in melon type in melon type of theSNP and plant compris- plant compris- recurrent nucleotide ing the donoring the donor parent, lacking position (nt) in fragment in fragment inhet- the introgres- the sequence homozygous form erozygous form sionfragment SNP_01 (nt 101 CC CT TT of SEQ ID NO 1) SNP_02 (nt 945 TT TG GGof SEQ ID NO 2) SNP_03 (nt 68 TT TC CC of SEQ ID NO 3) SNP_04 (nt 227 AAAG GG of SEQ ID NO 4) SNP_05 (nt 839 CC CT TT of SEQ ID NO 5) SNP_06 (nt445 AA AG GG of SEQ ID NO 6)

In one embodiment a cultivated melon plant (of the species C. melo) isprovided which comprises a recombinant chromosome 5, whereby therecombinant chromosome 5 comprises an introgression fragment thatconfers ToLCNDV resistance onto the melon plant when present inhomozygous or heterozygous form and wherein the introgression fragmentis from a wild donor of the species C. melo. In one aspect theintrogression fragment comprises the SNP donor genotype for one or moreor all of SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 and or SNP_06. In oneembodiment the introgression fragment comprises the donor SNP genotypefor at least SNP_03 and/or SNP_04. The SNP genotype of the donor may bepresent in homozygous form (if the introgression fragment is inhomozygous form) or in heterozygous form (if the introgression fragmentis in heterozygous form). So, for example, the plant or plant cell orplant part may comprise the TT or TC genotype for SNP_03 at nucleotide68 of SEQ ID NO: 3. Thus, the plant, plant cell, or plant part maycomprise SEQ ID NO: 3, or a sequence comprising at least 95%, 97%, or98% sequence identity to SEQ ID NO: 3, in homozygous form or inheterozygous form.

In one embodiment cultivated melon plants or cells of these plants areprovided which comprise an introgression fragment from a wild donor onchromosome 5, which introgression fragment confers the ToLCNDVresistance, whereby the introgression fragment lies in-between SNP_01and SNP_06 (or in-between SEQ ID NO:1 and SEQ ID NO:6, or a sequencecomprising at least 90%, or at least 95%, or at least 97% or 98%sequence identity to SEQ ID NO: 1 or to SEQ ID NO 6); or whereby theintrogression fragment lies in-between SNP_01 and SNP_05 (or in-betweenSEQ ID NO: 1 and SEQ ID NO:5, or a sequence comprising at least 90%, orat least 95%, or at least 97% or 98% sequence identity to SEQ ID NO: 1or to SEQ ID NO 5).

In one aspect the cultivated melon plants or cells of these plantscomprise an introgression fragment from a wild donor on chromosome 5,which introgression fragment confers the ToLCNDV resistance, whereby theintrogression fragment lies in-between SNP_01 and SNP_06 (or in-betweenSEQ ID NO:1 and SEQ ID NO: 6, or a sequence comprising at least 90%, orat least 95%, or at least 97% or 98% sequence identity to SEQ ID NO: 1,or to SEQ ID NO 6), or the fragment lies in-between SNP_01 and SNP_05(or in-between SEQ ID NO:1 and SEQ ID NO:5, or a sequence comprising atleast 90%, or at least 95%, or at least 97% or 98% sequence identity toSEQ ID NO: 1 or to SEQ ID NO 5), and whereby the introgression fragmentcomprises a Thymine (T) at nucleotide 68 of SEQ ID NO 3 or at theequivalent nucleotide of a sequence comprising substantial sequenceidentity to SEQ ID NO: 3.

In a further aspect the introgression fragment optionally furthercomprises an Adenine (A) at nucleotide 227 of SEQ ID NO 4 or at theequivalent nucleotide of a sequence comprising substantial sequenceidentity to SEQ ID NO: 4.

In a further aspect the introgression fragment optionally furthercomprises the donor nucleotide of SNP_02 and/or SNP_05. Thus, theintrogression fragment may comprise the donor genotype for SNP_03,SNP_04 and SNP_05; or for SNP_03 and SNP_02; or for SNP_03 and SNP_02and SNP_04; or for SNP_03, SNP_02, SNP_04 and SNP_05.

In another aspect the introgression fragment optionally furthercomprises the donor nucleotide of SNP_02 and/or SNP_01. Thus, theintrogression fragment may comprise the donor genotype for SNP_03 andSNP_02; or for SNP_03, SNP_02 and SNP_01; and optionally also forSNP_04, and further optionally also for SNP_05, and further optionallyfor SNP_06.

In yet another aspect the introgression fragment optionally furthercomprises the donor nucleotide of SNP_05 and/or SNP_06.

Thus in one aspect, the cultivated melon plants or cells of these plantscomprise an introgression fragment from a wild donor on chromosome 5,which introgression fragment confers the ToLCNDV resistance, whereby theintrogression fragment lies in-between SNP_01 and SNP_06 (or in-betweenSEQ ID NO:1 and SEQ ID NO:6, or a sequence comprising at least 90%, orat least 95%, or at least 97% or 98% sequence identity to SEQ ID NO: 1or to SEQ ID NO 6) and whereby the introgression fragment comprises theToLCNDV resistant SNP genotype for one or more or all of SNP_01, SNP_02,SNP_03, SNP_04, SNP_05, and SNP_06. In one aspect, the fragmentcomprises the donor SNP genotype for SNP_03 and/or SNP_04.

Thus in one aspect, the cultivated melon plants or cells of these plantscomprise an introgression fragment from a wild donor on chromosome 5,which introgression fragment confers the ToLCNDV resistance, whereby theintrogression fragment lies in-between SNP_01 and SNP_05 (or in-betweenSEQ ID NO: 1 and SEQ ID NO:5, or a sequence comprising at least 90%, orat least 95%, or at least 97% or 98% sequence identity to SEQ ID NO: 1or to SEQ ID NO 5) and whereby the introgression fragment comprises theToLCNDV resistant SNP genotype for one or more or all of SNP_01, SNP_02,SNP_03, SNP_04 and SNP_05, in one aspect, the fragment comprises thedonor SNP genotype for SNP_03 and/or SNP_04. Optionally the resistantSNP genotype is also present for SNP_06.

Thus another aspect, the cultivated melon plants or cells of theseplants comprise an introgression fragment from a wild donor onchromosome 5, which introgression fragment confers the ToLCNDVresistance, whereby the introgression fragment lies in-between SNP_03and SNP_04 (or in-between SEQ ID NO:3 and SEQ ID NO:4, or a sequencecomprising at least 90%, or at least 95%, or at least 97% or 98%sequence identity to SEQ ID NO: 3 or to SEQ ID NO 4) and whereby theintrogression fragment optionally comprises the ToLCNDV resistant SNPgenotype for SNP_03 and/or SNP_04. Optionally the donor genotype is alsopresent for one or more or all of SNP markers selected from SNP_01,SNP_02, SNP_05 and SNP_06.

In a further aspect, the cultivated melon plants or cells of theseplants comprise an introgression fragment from a wild donor onchromosome 5, which introgression fragment confers the ToLCNDVresistance, whereby the introgression fragment lies in-between SNP_02and SNP_04 (or in-between SEQ ID NO:2 and SEQ ID NO:4, or a sequencecomprising at least 90%, or at least 95%, or at least 97% or 98%sequence identity to SEQ ID NO: 2 or to SEQ ID NO 4) and whereby theintrogression fragment optionally comprises the ToLCNDV resistant SNPgenotype for SNP_02 and/or SNP_03 and/or SNP_04. Optionally the donorgenotype is also present for one or more or all of SNP markers selectedfrom SNP_01, SNP_05 and SNP_06.

Plants comprising plant cells according to the invention are anotherembodiment of the invention.

The melon plant according to the invention may be an inbred line, anopen pollinated variety (OP) or an F1 hybrid, in one aspect the F1hybrid comprises the introgression fragment in heterozygous form, i.e.produced by crossing two inbred parent lines, one of which possesses theintrogression fragment (preferably in homozygous form, although notnecessarily) and collecting the F1 hybrid seeds from said cross. The F1hybrid may also comprise the introgression fragment in homozygous form,i.e. produced by crossing two inbred parent lines, each comprising theintrogression fragment in homozygous or heterozygous form.

The melon plant according to the invention may be of any type.Preferably it has good agronomic and good fruit quality characteristics,such as large average fruit size (at least 500 g, 600 g, 700 g, 800 g,900 g, 1000 g or more), high average brix of the fruits (e.g. an averagerefractometer % total soluble solids of at least 10%, 12%, 14%, 16%, 18%or more), many fruits being produced per plant, firm fruit flesh, etc.

Also other resistances may be introduced into the melon plants of theinvention, such as resistance to one or more of the following diseases:Bacterial Wilt, Root Rot, Crown Blight, Melon Rust, Powdery Mildew,Verticillum Wilt, Sulphur Bum, Scab, Watermelon Mosaic, Downy Mildew,Fusarium oxysporum fsp. melonis (Fom) race 0, Fusarium oxysporum fsp.melonis (Fom) race 1, Fusarium oxysporum fsp. melonis (Fom) race 2,Fusarium oxysporum fsp. melonis (Fom) race 1.2, Fusarium Wilt

R2, Root Knot (Nematode), Anthracnose, Cucumber Mosaic, and SquashMosaic, and/or resistance to one or more of the following pests: Aphidresistance, Pickle Worm, Darkling Ground Beetle, Banded Cucumber Beetle,Mite, Western Spotted Cucumber Beetle, Melon Leafhopper, Melon Worm,Western Striped Cucumber Beetle or Melon Leafminer. Other resistancegenes, against pathogenic viruses, fungi, bacteria or pests may also beintroduced.

A specific aspect of the invention concerns plants or plant cellscomprising an introgression fragment according to the invention whichintrogression fragment is obtainable from seeds deposited under NCIMB42585 or from progeny thereof. The seeds deposited are cultivated melonplants of the BC4S4 generation comprising the introgression fragment inhomozygous form, with the donor nucleotide being present in homozygousform for SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 and SNP_06. The TolCNDVresistance is most likely located in-between SNP_03 and SNP_04, whichmeans that the size of the donor introgression can be reduced, byselecting recombinants having smaller introgression fragment sizes. Soplants comprising sob-fragments of the introgression fragment (whereinsaid sub-fragments still confer TolCNDV resistance), comprising thedonor SNP for SNP_03 and/or SNP_04, but having the SNP genotype of therecurrent parent for one or more or all of the other SNPs can begenerated in ways known to the skilled person.

Whether a plant comprises the ToLCNDV resistance from the depositedseeds (either an introgression fragment of the same size or asub-fragment thereof) can be determined by various methods, such assequencing and comparing the sequences of the introgression fragmentsand recombination sites.

Melon plants and plant parts (such as leaves, stems, roots, fruits,pollen, flowers, etc.) comprising melon plant cells according to theinvention are also an embodiment of the invention. Likewise seals fromwhich such plants can be grown are encompassed herein, as well as partsof such seeds (e.g. cells or tissues of the seeds such as the seed coat,embryo, etc.).

A further aspect of the present invention concerns melon seedscomprising an introgression fragment from chromosome 5 of a ToLCNDVresistant donor plant, wherein the introgression fragment comprises thesequence of the ToLCNDV resistant donor melon plant in-between SNP_01and SNP_06, in-between SNP_01 and SNP_05, in-between SNP_05 and SNP_04,in-between SNP_02 and SNP_06, in-between SNP_02 and SNP_05, in-betweenSNP_02 and SNP_04, SNP_03 and SNP_06, in-between SNP_03 and SNP_05 orin-between. SNP_03 and SNP_04. In a preferred embodiment of theinvention the seeds comprise an introgression fragment from chromosome 5of a ToLCNDV resistant donor plant, wherein the introgression fragmentcomprises the sequence of the ToLCNDV resistant donor melon plantin-between SNP_01 and SNP_06, more preferably in-between SNP_05 andSNP_05, even more preferably in-between SNP_02 and SNP_05, furthermorepreferably in-between SNP_02 and SNP_04 and most preferably in-betweenSNP_03 and SNP_04.

Another embodiment of the invention concerns melon seeds obtainable orobtained from plants according to the invention, or seeds comprisingplant cells according to the invention.

A further aspect of the present invention concerns melon plant fruitscomprising an introgression fragment from chromosome 5 of a ToLCNDVresistant donor plant, wherein the introgression fragment comprises thesequence of the ToLCNDV resistant donor melon plant in-between SNP_01and SNP_06, in-between SNP_01 and SNP_05, in-between SNP_01 and SNP_04,in-between SNP_02 and SNP_06, in-between SNP_02 and SNP_05, in-betweenSNP_02 and SNP_04, SNP_03 and SNP_06, in-between SNP_03 and SNP_05 orin-between SNP_03 and SNP_04. In a preferred embodiment of the inventionthe seeds comprise an introgression fragment from chromosome 5 of aToLCNDV resistant donor plant, wherein the introgression fragmentcomprises the sequence of the ToLCNDV resistant donor melon plantin-between SNP_01 and SNP_06, more preferably in-between SNP_01 andSNP_05, even more preferably in-between SNP_02 and SNP_05, furthermorepreferably in-between SNP_02 and SNP_04 and most preferably in-betweenSNP_03 and SNP_04.

Another embodiment of the invention concerns melon fruits obtainable orobtained from plants according to the invention, or fruits comprisingplant cells according to the invention.

Preferably melon fruits according to the invention are characterized inthat they comprise an introgression fragment from chromosome 5 of aToLCNDV resistant donor plant comprising the sequence of the donor plantin-between SNP_01 and SNP_06, in-between SNP_01 and SNP_05, in-betweenSNP_01 and SNP_04, in-between SNP_02 and SNP_06, in-between SNP_02 andSNP_05, in-between SNP_02 and SNP_04, in-between SNP_03 and SNP_06,in-between SNP_03 and SNP_05 or preferably in-between SNP_03 and SNP_04in heterozygous or homozygous state.

The preferred and further embodiments described herein for melon plantcells or melon plants according to the invention are applicable to alsorepresent preferred and further embodiments of the melon fruits of melonplants according to the invention, accordingly.

A further aspect of the present invention concerns melon plantpropagation material comprising an introgression fragment frontchromosome 5 of a ToLCNDV resistant donor plant, wherein theintrogression fragment comprises the sequence of the ToLCNDV resistantdonor melon plant in-between SNP_01 and SNP_06, in-between SNP_01 andSNP_05, in-between SNP_01 and SNP_04, in-between SNP_02 and SNP_06,in-between SNP_02 and SNP_05, in-between SNP_02 and SNP_04, SNP_03 andSNP_06, in-between SNP_03 and SNP_05 or in-between SNP_03 and SNP_04. Ina preferred embodiment of the invention the propagation materialcomprises an introgression fragment from chromosome 5 of a ToLCNDVresistant donor plant, wherein the introgression fragment comprises thesequence of the ToLCNDV resistant donor melon plant in-between SNP_01and SNP_06, more preferably in-between SNP_01 and SNP_05, even morepreferably in-between SNP_02 and SNP_05, furthermore preferablyin-between SNP_02 and SNP_04 and most preferably in-between SNP_03 andSNP_04.

Another embodiment of the invention concerns melon plant propagationmaterial obtainable or obtained from plants according to the invention,or melon plant propagation material comprising plant cells according tothe invention.

The preferred and further embodiments described herein for plant cellsor plants according to the invention are applicable to also representpreferred and further embodiments of the propagation material of melonplants according to the invention, accordingly.

The term “propagation material” comprises those components of the plantwhich are suitable for generating progeny via the vegetative (agamic) orgenerative (gamic, sexual) route. Suitable for vegetative propagationare, for example, cuttings, in vitro tissue, cell, protoplast, embryo orcallus cultures, micropropagation methods, rhizomes or tubers. Otherpropagation material includes, for example, fruits, seeds, seedling,being homozygous or heterozygous for an chromosome 5 introgressionfragment conferring ToLCNDV resistance etc. The propagation material inone aspect takes the form of cuttings which are propagated by graftingto another rootstock or in vitro tissue culture material, in particularembryo cultures, in particular preferred is propagation material in theform of in vitro tissue culture material, particularly in vitro embryocultures.

In one aspect non-propagating plant ceils comprising the recombinantchromosome 5 described herein are provided. In one aspect suchnon-propagating plant cells may however be part of a melon plant ormelon plant part.

A further embodiment of the invention concerns a method for producing aToLCNDV resistant melon plant comprising the following steps

-   -   a) Selecting a ToLCNDV resistant donor plant    -   b) Crossing the donor plant selected in step a) with a recurrent        plant sensitive to ToLCNDV    -   c) Obtaining seeds from the plants crossed in step b) and        optionally    -   d) Verifying if the plants grown from the seeds obtained in        step c) are resistant to ToLCNDV and/or comprise one or more of        the SNPs from the donor plant selected from the group of SNP_01,        SNP_02, SNP_03, SNP_04, SNP_05 and SNP_06.

A ToLCNDV resistant donor plant in step a) in the method for producing aToLCNDV resistant melon plant according to the invention can be selectedby infection of melon plants with ToLCNDV and determining the level ofsymptoms of ToLCNDV infected melon plants as described elsewhere herein.The same is applicable for verification in steps b) and c) of the methodfor producing a ToLCNDV resistant melon, plant according to theinvention, if a plant is ToLCNDV sensitive or resistant, respectively.

In a preferred embodiment of the method for producing a ToLCNDVresistant melon plant according to the invention the ToLCNDV resistantdonor plant in step a) comprises a fragment on chromosome 5 conferringToLCNDV resistance, the fragment comprising the sequence of the donorplant in-between SNP_01 and SNP_06, in-between SNP_01 and SNP_05,in-between SNP_01 and SNP_04, in-between SNP_02 and SNP_06, in-betweenSNP_02 and SNP_05, in-between SNP_02 and SNP_04, in-between SNP_03 andSNP_06, in-between SNP_03 and SNP_05 or in-between SNP_03 and SNP_04.Most preferably the fragment on chromosome 5 conferring ToLCNDVresistance comprises the sequence in-between SNP_03 and SNP_04. In aspecifically preferred embodiment of the invention, the method forproducing a ToLCNDV resistant melon plant according to the invention theToLCNDV resistant donor plant in step a) comprises the fragment ofchromosome 5 conferring ToLCNDV resistance comprising the sequencein-between SNP_01 and SNP_06, in-between SNP_01 and SNP_05, in-betweenSNP_01 and SNP_04, in-between SNP_02 and SNP_06, in-between SNP_02 andSNP_05, in-between SNP_02 and SNP_04, in-between SNP_03 and SNP_06,in-between SNP_03 and SNP_05 or preferably in-between SNP_03 and SNP_04as found in the seeds deposited under NCIMB 42585.

In a preferred embodiment of the invention the method for producing aToLCNDV resistant melon plant according to the invention is used forproducing a plant according to the invention. The preferred and furtherembodiments as described herein for the plants according to theinvention are applicable accordingly to the method for producing aToLCNDV resistant melon plant according to the invention.

Plants obtainable or obtained by a method for producing a ToLCNDVresistant melon plant according to the invention are also an embodimentof the invention.

A further embodiment of the invention concerns methods for producingmelon seeds comprising the following steps

-   -   a) growing a melon plant comprising at least one chromosome 5        having an introgression fragment from chromosome 5 of a ToLCNDV        resistant donor plant, the introgression fragment comprising the        sequence of the donor plant in-between SNP_03 and SNP_04.    -   b) harvesting the fruits of the melon plants grown in step a)    -   c) collecting the seeds from the fruits obtained in step b).

In a preferred embodiment of the invention the melon plants of steps a)of the method for producing melon seeds according to the invention hasthe specific characteristics described as preferred and furtherembodiments of the plants according to the invention. The preferred andfurther embodiments as described herein for the plants according to theinvention are applicable accordingly to the method for producing ahybrid melon seed according to the invention.

Seeds obtainable the method for producing melon seeds according to theinvention are also an embodiment of the invention.

Another embodiment of the invention concerns methods for producinghybrid melon seeds comprising the following steps

-   -   a) providing a first inbred melon plant comprising at least one        chromosome 5 having an introgression fragment from chromosome 5        of a ToLCNDV resistant donor plant, the introgression fragment        comprising the sequence of the donor plant in-between SNP_03 and        SNP_04.    -   b) providing a second inbred melon plan t with or without a        chromosome 5 having an introgression fragment from chromosome 5        of a ToLCNDV resistant donor plant, the introgression fragment        comprising the sequence of the donor plant in-between SNP_03 and        SNP_04.    -   c) crossing the plant provided in step a) with the plant        provided in step b)    -   d) selecting seeds obtained from the cross of step c).

“Inbred plant” or “inbred line” shall mean in connection with thepresent invention plants which have undergone several generations ofselling and are highly uniform in respect to their genetic setup andphenotypic appearance.

In a preferred embodiment of the invention the inbred lines of steps a)and b) of the method for producing hybrid melon seeds according to theinvention has the specific characteristics described as preferred andfurther embodiments of the plants according to the invention. Thepreferred and further embodiments as described herein for the plantsaccording to the invention are applicable accordingly to the method forproducing a hybrid melon seed according to the invention.

Hybrid seeds obtainable or obtained by the method for producing hybridmelon seeds according to the invention are also an embodiment of theinvention.

A further embodiment of the present invention are methods for producinga melon fruit comprising the following step

-   -   a) growing a plant comprising at least one chromosome 5 having        an introgression fragment from chromosome 5 of a ToLCNDV        resistant donor plant, the introgression fragment comprising the        sequence of the donor plant in-between SNP_03 and SNP_04,    -   b) harvesting the fruits produced by the plants grown in step        a).

The term “fruit” in its botanical meaning is commonly understood to be aseed bearing structure developed from the ovary of angiosperm flowers.

Melon fruits obtainable or obtained by a method for producing a melonfruit according to the invention are also an embodiment of theinvention.

Melon donor plants being resistant to ToLCNDV can be identified with theaid of the SNP markers, in particular one or more or all of SNP_01,SNP_02, SNP_03, SNP_04, SNP_05 and SNP_06 disclosed herein. The presentinvention therefore for the first time enables a person skilled in theart to identify donor plants from which an introgression fragmentconferring ToLCNDV resistance to melon plants can be transferred intorecurrent melon plants.

A further embodiment of the invention therefore pertains the use of oneor more or all of SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 or SNP_06 foridentification of a ToLCNDV resistant melon plant or parts thereof (suchas cells, fruits, leaves). Preferably the use pertains theidentification of ToLCNDV resistant donor melon plants and/or recurrentmelon plants, but also to the identification of breeding lines,cultivars or varieties containing a recombinant chromosome 5, e.g. therecombinant chromosome 5 derived from the seeds deposited herein,optionally comprising a subfragment of the introgression fragmentpresent in the seeds deposited.

Another embodiment is the use of one or more or all of SNP_01, SNP_02,SNP_03, SNP_04, SNP_05 or SNP_06 for introgression of ToLCNDV resistanceinto a TolCNDV susceptible melon plant, especially a cultivated melonline or variety.

Also an embodiment of the invention is the use of one or more or all ofSNP_01, SNP_02, SNP_03, SNP_04, SNP_05 or SNP_06 in breeding ToLCNDVresistant melon plants.

Also provided is a method of screening plants or plant material or DNAderived therefrom for the presence of a fragment on chromosome 5conferring TolCNDV resistance. The method comprises the steps of:

-   -   screening the genomic DNA for the SNP genotype of one or more or        all of SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 and SNP_06;    -   and optionally selecting plants or plant material which comprise        the resistant donor genotype of one or more or all of SNP_01,        SNP_02, SNP_03, SNP_04, SNP_05 and SNP_06.

Also provided is a method for producing a cultivated C. melo plantcomprising an introgression fragment on chromosome 5, wherein saidintrogression fragment comprises a ToLCNDV QTL, comprising:

-   -   crossing a first cultivated melon plant being susceptible to        ToLCNDV with a second wild melon plant being resistant to        ToLCNDV, wherein said second melon plant comprises the CC or CT        genotype for SNP_01 in SEQ ID NO: 1, and/or the TT or TG        genotype for SNP_02 in SEQ ID NO: 2, and/or the TT or TC        genotype for the SNP_03 in SEQ ID NO: 3 and/or the AA or AG        genotype for the SNP_04 in SEQ ID NO: 4, and/or the CC or CT        genotype for SNP_05 in SEQ ID NO: 5, and/or the AA or AG        genotype for SNP_06 in SEQ ID NO: 6;    -   collecting F1 seeds front said cross and backcrossing an F1        plant to the first melon plant to produce a backcross (BC1)        population, or selfing said F1 plants one or more times to        produce an F2 or F3 population, and optionally selling the        backcross population to produce a BC1S1 population,    -   wherein said F2, F3, BC1 or BC1S1 plant comprises the CC or CT        genotype for SNP_01 in SEQ ID NO: 1, and/or the 1′T or TG        genotype for SNP_02 in SEQ ID NO: 2, and/or the TT or TC        genotype for the SNP_03 in SEQ ID NO: 3 and or the AA or AG        genotype for the SNP_04 in SEQ ID NO: 4, and/or the CC or CT        genotype for SNP_05 in SEQ ID NO: 5, and/or the AA or AG        genotype for SNP_06 in SEQ ID NO: 6.

Also provided is a method for identifying or detecting a cultivated C.melo plant comprising an introgression fragment on chromosome 5, whereinsaid introgression fragment comprises a ToLCNDV-resistance allele,comprising:

-   -   screening a Cucumis melo plant using a molecular marker assay        which detects at least one of SNP marker selected from the group        consisting of: SNP_01 in SEQ ID NO: 1, SNP_02 in SEQ ID NO: 2,        SNP_03 in SEQ ID NO; 3, SNP_04 in SEQ ID NO: 4, SNP_05 in SEQ ID        NO: 5 and/or SNP_06 in SEQ ID NO: 6; and    -   identifying and/or selecting a plant comprising the CC or CT        genotype for SNP_01 in SEQ ID NO: 1, and/or the TT or TG        genotype for SNP_02 in SEQ ID NO: 2, and/or the TT or TC        genotype for the SNP_03 in SEQ ID NO: 3 and/or the AA or AG        genotype for the SNP_04 in SEQ ID NO: 4, and/or the CC or CT        genotype for SNP_05 in SEQ ID NO: 5, and/or the AA or AG        genotype for SNP_06 in SEQ ID NO: 6.

A method of producing C. melo F1 hybrid plants comprising a ToLCNDVresistance phenotype is provided comprising:

-   -   crossing a first inbred melon plant comprising at least one        recombinant chromosome 5, the recombinant chromosome 5        comprising an introgression fragment that confers ToLCNDV        resistance onto the first inbred melon plant when present in        homozygous or heterozygous form and wherein said introgression        fragment is from a wild plant of the species Cucumis melo, with        a second inbred melon plant with or without said at least one        recombinant chromosome 5 and    -   collecting F1 hybrid seeds from said cross.

Further encompassed is a method for producing a melon plant comprisingToLCNDV resistance on chromosome 5, said method comprising:

-   -   a) screening a wild melon accession or several wild melon        accessions using a molecular marker assay which detects at least        one of SNP marker selected from the group consisting of: SNP_01        in SEQ ID NO: 1, SNP_02 in SEQ ID NO: 2, SNP_03 in SEQ ID NO: 3,        SNP_04 in SEQ ID NO: 4, SNP_05 in SEQ ID NO: 5 and/or SNP_06 in        SEQ ID NO: 6    -   b) identifying and/or selecting a wild melon plant comprising        the CC or CT genotype for SNP_01 in SEQ ID NO; 1, and/or the TT        or TG genotype for SNP_02 in SEQ ID NO: 2, and/or the TT or TC        genotype for the SNP_03 in SEQ ID NO: 3 and/or the AA or AG        genotype for the SNP_04 in SEQ ID NO: 4, and/or the CC or CT        genotype for SNP_05 in SEQ ID NO: 5, and or the AA or AG        genotype for SNP_06 in SEQ ID NO: 6.    -   c) optionally confirming ToLCNDV resistance in a resistance        assay;    -   d) and optionally introgressing said ToLCNDV resistance from        said wild accession into a cultivated melon plant.

A Cucumis melo plant, or part thereof, is provided comprising arecombinant chromosome 5, the recombinant chromosome 5 comprising anintrogression fragment that confers ToLCNDV resistance onto the Cucumismelo plant when, present in homozygous or heterozygous form and whereinsaid introgression fragment comprises one or more or all of the SingleNucleotide Polymorphism (SNP) markers of the group:

the CC or CT genotype for SNP_01 in SEQ ID NO: 1, the TT or TG genotypefor SNP_02 in SEQ ID NO: 2, the TT or TC genotype for the SNP_03 in SEQID NO: 3, the AA or AG genotype for the SNP_04 in SEQ ID NO: 4, the CCor CT genotype for SNP_05 in SEQ ID NO: 5, and/or the AA or AG genotypefor SNP_06 in SEQ ID NO: 6,

and wherein said introgression fragment is from a wild plant of thespecies Cucumis melo, said wild plant having an average ToLCNDV diseasescore of at least 7.0 on a scale of 1=dead plant to 9=no symptoms.

In one aspect the cultivated melon plant comprises at least the TT or TCgenotype for the SNP_03 in SEQ ID NO: 3 and/or the AA or AG genotype forthe SNP_04 in SEQ ID NO: 4.

In one aspect the ToLCNDV resistance QTL or the introgression fragmentcomprising the QTL is the obtainable from/can be obtained from/is aspresent in seeds of which a representative sample has been depositedunder Accession Number NCIMB42585 or progeny thereof (whereby theprogeny retain the ToLCNDV resistance).

Seed Deposit Information

A representative sample of seeds of a cultivated melon, designatedCucumis melo TOLCHR5, comprising an introgression fragment comprisingTolCNDV resistance introgressed on chromosome 5 (backcross 4, selfing 4generation, BC4S4), was deposited by Nunhems B. V. on 6 Jun. 2016 at theNCIMB Ltd. (Ferguson Building, Craibstone Estate, Bucksburn Aberdeen,Scotland AB21 9YA, UK) according to the Budapest Treaty, under theExpert Solution (EPC 2000, Rule 32 (1)). Seeds were given the followingdeposit numbers NCIMB 42585.

The Applicant requests that samples of the biological material and anymaterial derived therefrom be only released to a designated Expert inaccordance with Rule 32 (1) EPC or related legislation of countries ortreaties having similar rules and regulation, until the mention of thegrant of the patent, or for 20 years from the date of filing if theapplication is refused, withdrawn or deemed to be withdrawn.

Access to the deposit will be available during the pendency of thisapplication to persons determined by the Director of the U.S. PatentOffice to be entitled thereto upon request. Subject to 37 C.F.R. §1.808(b), all restrictions imposed by the depositor on the availabilityto the public of the deposited material will be irrevocably removed uponthe granting of the patent. The deposit will be maintained for a periodof 30 years, or 5 years after the most recent request, or for theenforceable life of the patent whichever is longer, and will be replacedif it ever becomes nonviable during that period. Applicant does notwaive any rights granted under this patent on this application or underthe Plant Variety Protection Act (7 USC 2321 et seq.).

Description of Sequences

Characters other than G (guanine), A (adenine), T (thymine) and C(cytosine) have the following meaning in the SEQ ID NOs shown in thesequence listing:

-   R: G or A-   Y: T or C-   M: A or C-   K: G or T-   S: G or C-   W: A or T-   H: A or C or T-   B: G or T or C-   V: G or C or A-   D: G or A or T-   N: G or A or T or C

In SEQ ID NO: 1 to SEQ ID NO: 6 the SNP nucleotide of the resistantdonor is shown in bold and underlined.

SEQ ID NO 1: Sequence of the ToLCNDV resistant donor plant comprising SNP_01.SEQ ID NO: 1:agctggtgca aagctggcat tcaaatcgaa tgaagaaata gcagtacaag tgaagtcaat   60tccactagat gaagtaatcc cggattcaga acgagtgctt  c taataaaaa tcgatgttca  120aggctgggaa tatcatgtgc taaaaggggc aaagagaatt ttgtcaagga agggcactga  180agctccatat ctcatctatg  200SEQ ID NO 2: Sequence of the ToLCNDV resistant donor plant comprising SNP_02.SEQ ID NO: 2ttgtaatcat ggccattgcc tgatytgcag aatgtggcgt cttgattggg cacggtctgg   60atgaagttac tccaaagctt gtggaagtta atgggttctt ctcttcaccr tctgrtctcc  120ttcccatcga caatgtggtt tcttcttcaa acacaaaaga aacccctgtt gaagctgtag  180aagaaagctg tgagttcttt tctccactca gttctaggtt tagtttttta tttgtttttt  240ttttcttttt tcctttttac ttgctttgag ctgtaggaca caacacaaaa tgataagaac  300aaaaatcaat ctatgttatt gttgtcattg tctcgcctgc ctcttcaagg taactaggct  360ataacactga tccaaaatgt ataatcttta ttgctaagta acttatattg caaagattgg  420gatggaatag gagcataggt tgcatctaat agttttgtcc aactttggtt atagtttcag  480ccagtttgtt cagatgaaat tcttagtgat tatcactaat ggaattgttt ttataaatgt  540tgtaggtgtt gaaagaagtg aagaatatga aaaggaaagc aggggaaccg agaaggctga  600aatcttaccg acaaaagcaa catctgaagc aggttctgaa gtccaacctg tttccagtga  660ttctgctcag atggtaccca atatgttgga gctcggtgat gcttataagc tagctgtagg  720tgctagagga ggaagacaat tgtctggcaa gcttttggaa caatggatyg ggaaggaatc  780ttcaaaagtt agtgaagatc tgaagcttct cttgacacaa ctctcattta atcgtttgaa  840tgaccaatca cgggagatga gtccaaggct gtccgtaaat ggagacgagg tgaggaactt  900tgattacttg agygctgttg ggatgcaaat gctacaaaaa aggw t ttcgc ttgaaagaaa  960tgagtccggt gtagaatctt tagatggaag cataattagt gaaatcgatg gggaaaacat 1020ggctgatagg ttgaaacgac agattgagta tgataagaag gttmtgartt ctttatacaa 1080ggaattggag gaagaaagaa atgcatccgc aattgctgca aatcaggcaa tggccatgat 1140tacaa 1145SEQ ID NO 3: Sequence of the ToLCNDV resistant donor plant comprising SNP_03.SEQ ID NO 3agacggcgta catgcagcat tctgttaccc cgcgatgccc agcgatgaaa caactccttt   60gcatcct t at ggtccagagg ttccagatca tacgactcac caaatgcagg aaacttaaat  120ctagaggtga ccaaaatttt gcagttgggt aatcgggaga acttttca  168SEQ ID NO 4: Sequence of the ToLCNDV resistant donor plant comprising SNP_04.SEQ ID NO 4ggagkcttca tccgtcttta ccttcctcac ttttttacct aaccaagtta cgtcttgtag   60gttgcaagat aacaaatttg gatttcttag aaacaattgt ttatgttgcc ccttcrttga  120aagagttgga cttgtccgaa aacaactttt gtagamtacc ctcrtgtatt attaatttta  180aatccctgaa atatctttat acaatggatt gtgagttgct ygaaga a att tcaaaggttc  240cagaaggtgt aatttgtacg agtgccgcag gatgcaaatc attggctaga tttcccgaca  300acttagctga tttcatatct tgtggtaatt ctgcggtgcg taccatatct ctttctcatg  360acttcaccat tatctctagc tcatgtatat ttaatttcat tcatataata tatattactt  420ataactattt actgatctca tggtgcagga atgttgtaaa ggtggatg  468SEQ ID NO 5: Sequence of the ToLCNDV resistant donor plant comprising SNP_05.SEQ ID NO: 5tgcaggtagt caaaccaatt gataatattt gctcaccatc aaaattggta accatagaca   60tagaytgacc aagcccaggc aaaacggcat catctttttg ttttctatca ttactattct  120gattacccaa aagaaatctt ctttttgtac ttggagagaa ataatccccg tctgcacatt  180gcatggaagg tgtgctgtca tgagcatcag cattacacac accagaatta gatttcttgc  240tagccaagat ttcacaacca ccaagagaag caggacctac gcctggggtc tctgcctyag  300aaagccaags atcaaagcac acttgtgaag gacctgaaac tgtctcagat ccagggattt  360ctcctacatt gggtaagcta gcctgttcaa agccatcaca cacatctaca agcggcagtc  420taccatacat ggaactatta gaataagcag gcttattttc ataaaacaca ttaacgcttg  480catttggatt agttaaacat ggcatcaaat gggaagaatc ccgaaatctc aagctgttct  540cattggaccg cagcactcca gaattcaagt ctcttgacca tacttttttt tgcctgctat  600caacagtaaa attttgagct ttaccatatc cacagtcaag tggctcattt cgagagttac  660gcttcctcct cattaaatca cktacagaac ggccttccct ggagttatgt gtgcctgtac  720ctgcatttcg taatgtaaca tccacctgcc ctccatcttc acttgaaaag caactaatga  780actcagaatc tttggtcgaa cttgtccata tgtcaggcct gcaagaattg acacacgt c g  840aatctgtatt cactttctca gcctcatgaa aaggtaaaga tccccaaaat gatttctctt  900tcttgtgact gcaagaacct gaatcaatgc catgcaataa tattgcaagt ttggaagatc  960tttcattctc aacattaaga tctctatcag gtgagttttc agtcgaccct ccagaagaag 1020agaacatatc atctgcakgt acgcagt 1047SEQ ID NO 6: Sequence of the ToLCNDV resistant donor plant comprising SNP_06.SEQ ID NO: 6gcgtacmtgc aggcaaagaa tggcacagta cagtaatggt agctcatctg ttcatcaagg   60agaatcaagc tgcctcagcc attcagtatc ggttccaccc ctgatcatct cctacaatga  120tcgcattcgt cctctccttg atgctgttga caagcttcgt cacctcatga tcatgagaga  180aggcatccaa ctgcctacca tagttgttgt tggtgatcag tcawccggta agtcaagtgt  240cctcgagtcg ttggctggga tcagcctacc tcgaggtcag ggcatctgca ccagggtccc  300tctgataatg aggctccaaa accatcctga tcccgaaccc gagcttgttt tggagtacaa  360tgggaaaaag atccacaccg acgaatcctt cattgctgaa gacatctgta cagctacaga  420ggagattgct ggcagtggca aagg a atatc gaaagcgcca ttgactttga ttgtgaagaa  480aaatggtgtt cctgatctta caatggttga tctccctgga attacragag tgcctgttaa  540agatcagcct gaagacattt atgaccaaat aaaagatata atcatggaac atatcaagcc  600agaagagagc atcatcttga atgtcttgtc tgcgacggtt gattttccaa cttgtgaatc  660gatacggatg tctcaaagtg tcgacaagac gggaatgaga acgttggcag ttgtgactaa  720gtctgacaag gcaccagaag gcctacacga gaaggtcacc rcggatgatg tcagtatcgg  780ccttggttat gtttgcgtta ggaaccgaat tggcaatgag acatatgagg aagctcgggt  840tgcagaagcc aaattgtttt caactcatcc tcttctctcc aaaattgaca aatctgttgt  900gggcattcca gtcttggctc agaagttggt gcaaattcaa gcaggtaccc aaactaattc  960ctgactcaaa agctaggttc cgttagataa ccattttgtt ttagaaaatc aagtttattt 1020tctctaaatm gtgtaccatg attttcatct ttcttaaata aaaaagttgm attcttwact 1080aaattttaaa agcaaaaaca agttttaata cttttttt 1118

-   SEQ ID NO 7: KASP-assay primer for FAM allele of SNP_01.-   SEQ ID NO 8: KASP-assay primer for VIC allele of SNP_01.-   SEQ ID NO 9: KASP-assay common primer for SNP_01.-   SEQ ID NO 10: KASP-assay primer for FAM allele of SNP_02.-   SEQ ID NO 11: KASP-assay primer for VIC allele of SNP_02.-   SEQ ID NO 12: KASP-assay common primer for SNP_02.-   SEQ ID NO 13: KASP-assay primer for FAM allele of SNP_03.-   SEQ ID NO 14: KASP-assay primer for VIC allele of SNP_03.-   SEQ ID NO 15: KASP-assay common primer for SNP_03.-   SEQ ID NO 16: KASP-assay primer for FAM allele of SNP_04.-   SEQ ID NO 17: KASP-assay primer for VIC allele of SNP_04.-   SEQ ID NO 18: KASP-assay common primer for SNP_04.-   SEQ ID NO 19: KASP-assay primer for FAM allele of SNP_05.-   SEQ ID NO 20: KASP-assay primer for VIC allele of SNP_05.-   SEQ ID NO 21: KASP-assay common primer for SNP_05.-   SEQ ID NO 22: KASP-assay primer for FAM allele of SNP_06.-   SEQ ID NO 23: KASP-assay primer for VIC allele of SNP_06.-   SEQ ID NO 24: KASP-assay common primer for SNP_06.

DESCRIPTION OF THE FIGURES

FIG. 1: Shown is a ToLCNDV sensitive recurrent plant (upper picture) anda recurrent plant into which ToLCNDV resistance was integrated (lowerpicture) by introgression of a fragment comprising the sequence of thedonor in-between SNP_01 and SNP_06. The picture was taken 25 days postinfection (dpi) with ToLCNDV by whitefly transmission.

FIG. 2: Shown are the symptom levels 35 days post infection (dpi) withToLCNDV by whitefly transmission of a donor plant being ToLCNDVresistant (Wild Donor), a recurrent plant (Recurrent) and a plantobtained after introgression of the ToLCNDV resistance from the donorplant into the recurrent plant (Introgression). The symptom levels weredetermined as described herein under “General Methods”.

GENERAL METHODS 1. Determination of Symptom Level on ToLCNDV InfectedPlants

1.1 Plants and Pathogens (Virus)

A melon plant (Cucumis melo) infecting strain of ToLCNDV is used forinfection of melon plants. In the present invention a ToLCNDV stainisolated in Murcia, Spain was used as inoculum.

1.2 ToLCNDV Propagation

The ToLCNDV inoculum source is maintained on living infected melonplants. It must be ensured, that pure virus isolates are used and thatneither the virus source, nor the whiteflies are contaminated with otherdiseases, in particular with other viruses (e.g. CGMMV, CYSDV, CYVYSqMV). For pre-multiplication of the ToLCNDV inoculum whiteflies(Bemisia tabaci) are led on ToLCNDV sensitive (susceptible), infectedmelon plants in an insect proven cage. Before infection of test plants,put ToLCNDV infected plants into an insect proven cage, releasewhiteflies to the same cage and allow the whiteflies to feed forapproximately 3 days on the ToLCNDV infected plants.

1.3 Inoculation of Plants to be Tested

For each genotype of melon plants to be analysed 14 plants were grownuntil the first true leaf is expanded (normally 12-15 days aftersowing), 12 of which were infected and 2 were mock infected. Also 12plants of susceptible varieties were included, in this experimentvariety Gandalf F1 (Hild Samen) and variety Vedantrais. The 12 plantsper genotype to be tested on ToLCNDV resistance were put into an insectproof cage, infected whiteflies obtained as described under 1.2 above,were released into the cage to infect the plants. It has to be ensuredthat at least 5-10 whiteflies are available for each test plant in thecage. Whiteflies and test plants are kept in the cage for approximately48 hours, before the whiteflies are eliminated with an appropriateinsecticide. Also two plants per genotype were mock infected, i.e. theywere treated in the same manner as the test plants apart from that, thewhiteflies used for infection were free of ToLCNDV.

1.4 Growing Infected Test Plants

Infected test plants obtained as described under 1.3 were transplantedto bigger pots, transferred into a greenhouse with cooling equipment.The plants were grown at approximately 18° C. night temperature andapproximately 25° C. day temperature in a timeframe of 14 to 16 hoursdaylight. The infected plants for each infected genotype were grown intwo replicates in two different plots, each of which comprises 6 ToLCNDVinfected plants and 1 mock infected plant. The plots are randomized inrespect to the growing area.

1.5. Scoring the Symptom Level of ToLCNDV Infection

The scoring of the symptom level may already be done approximately 15days post infection (dpi) with ToLCNDV but is preferably doneapproximately 30 days post infection (dpi) with ToLCNDV, or later. Incase plants are present which show recovery from the virus infection, afurther scoring of the symptom is done approximately 45 days postinfection (dpi) with ToLCNDV.

The following symptom levels are to be used according to the phenotypesindicated in the following:

Symptom level Observed phenotype 1 Dead plant 2 Severe mosaic andcurling, chlorosis and growth reduction. No recovery 3 Strong mosaic andcurling, chlorosis and growth reduction. No recovery 4 Curling andmosaic, chlorosis, no or mild growth reduction. No recovery 5 Curlinganti mosaic, chlorosis, no growth reduction. Slight recovery of theupper plant zone 6 Mild curling, mosaic and chlorosis, no growthreduction. Recovery of the upper middle plant 7 Mild curling, mosaic andchlorosis, no growth reduction. Symptoms appear only in the lower plantzone 8 Faint mosaic 9 No symptoms

1.6 Optional Additional Tests

It is recommended to use at least one genotype highly resistant toToLCNDV (symptom level 8-9) and one genotype highly sensitive to ToLCNDV(symptom level 1) in each experimental setup. It is further recommendedto also include a genotype being intermediate resistant to ToLCNDVinfection in each test setup. Best results are obtained when the justmentioned genotypes are included and the symptom levels of each genotypeis scored relative to the results obtained for the highly resistant,highly sensitive and intermediate resistant genotypes. These genotypesalso give a clear indication on the amount of infection of melon plantswith ToLCNDV by the whiteflies.

Furthermore, it is advisable to check infection and spreading of ToLCNDVin infected plants and control plants. This can be done by checking forthe presence and amount of virus DMA in upper parts of the plants. Asuitable way to cheek for the presence and amount of ToLCNDV DNA inupper plant parts is hybridization of plant material with a probehybridizing with the DNA of the ToLCNDV strain used. Varioushybridization techniques are well known in the art. A simple so calledDot Blot analysis is sufficient for obtaining valuable results. LikewisePCR or quantitative PCR techniques can be used.

EXAMPLES 1. Selection of ToLCNDV Resistant Donor Plants

The symptom level of wild accessions of melon plants were tested forToLCNDV resistance according to the test described under “GeneralMethods”. A wild donor plant was identified which has a high resistanceto ToLCNDV infection, having a resistance level of about 7 (as seenfurther below the average disease score was 7.4, while the susceptibleplant had an average score of 2.0).

2. Identification of Genomic Location of ToLCNDV Resistance

Three mapping populations were developed including the use of the donorplant obtained in Example 1 to map the position of the ToLCNDVresistance conferring fragment (QTL) in the genome of donor melon plant.

Analysts in these mapping populations revealed one major QTL associatedwith resistance, located on chromosome 5 and showing dominantinheritance patterns.

The magnitude of the detected fragment QTL and observed inheritancepatterns suggested a single locus dominant gene. From resistant materialthe inventors developed BC (back cross) lines to line map and furtherinvestigate resistance from the donor. Genotypic results in 10 advancedBC families developed through phenotypic selection showed >93% agreementwith individual phenotypes.

The markers identified during line mapping and their respectivepositions according to publicly known data from Diaz et al. (2015, MolBreeding 35, 188) is shown in the following Table:

Pseudo- SNP molecule SNP Marker ID position coordinates AI_13-H12CM3.5_scaffold00003 7996720 27276249 SNP_01 mME11320_kCM3.5_scaffold00003 7824960 SNP_02 mME43070_k CM3.5_scaffold000036950286 SNP_03 mME10621_k CM3.5_scaffold00003 6553350 SNP_04 mME50729_kCM3.5_scaffold00003 5785550 SNP_05 mME32395_k CM3.5_scaffold000035202092 CMGAAN144 CM3.5_scaffold00003 5123876 24403405 CMPSNP682CM3.5_scaffoldO0009 4703882 15854444 SNP_06 mME49184_kCM3.5_scaffold00009 2607437 CMPSNP460 CM3.5_scaffold00009 181610912966671

3. Development of KASP-Assay

A KASP-assay was developed for identifying the SNPs flanking the QTL.The SNPs associated with the QTL can be determined by use of thefollowing primers in a KASP-assay:

SNP FAM allele VIC allele Common Primer SNP_01 SEQ ID NO 7 SEQ ID NO 8SEQ ID NO 9 SNP_02 SEQ ID NO 10 SEQ ID NO 11 SEQ ID NO 12 SNP_03 SEQ IDNO 13 SEQ ID NO 14 SEQ ID NO 15 SNP_04 SEQ ID NO 16 SEQ ID NO 17 SEQ IDNO 18 SNP_05 SEQ ID NO 19 SEQ ID NO 20 SEQ ID NO 21 SNP_06 SEQ ID NO 22SEQ ID NO 23 SEQ ID NO 24

4. Introgression of ToLCNDV into a Cultivated Melon Plant

Backcrossing has been performed with cultivated melon plants andrecurrent melon plants having an increased resistance to ToLCNDV havebeen obtained. The presence of the QTL in these lines was established byuse of the KASP-assay sequences shown in Example 3. A representativeresult for the ToLCNDV symptom level achieved in those plants is givenin the following fable and in FIG. 2. The symptom levels have beendetermined 35 days post infection (dpi) according to the methoddescribed herein under “General Methods”.

AVG STDV Wild Donor 7.40 2.07 Recurrent 2.00 0.00 introgression 5.111.45 AVG: average value of tested genotype, STDV: standard deviation

Additional backcrossing is done to introgress the resistance in elitePiel de Sapo, Galia, Cantaloup and Charentais backgrounds. Genotypicresults in 10 advanced BC families developed through phenotypicselection show >93% agreement with individual phenotypes.

The invention claimed is:
 1. A cultivated melon plant cell of thespecies Cucumis melo comprising an introgression fragment fromchromosome 5 of a ToLCNDV resistant donor plant, wherein theintrogression fragment confers ToLCNDV resistance and comprises aThymine at nucleotide 68 of SEQ ID NO: 3 and/or an Adenine at nucleotide227 of SEQ ID NO: 4, and wherein the introgression fragment isobtainable from seeds of which a representative sample has beendeposited under Accession Number NCIMB
 42585. 2. The cultivated melonplant cell according to claim 1, wherein the introgression fragmentcomprises a Cytosine at nucleotide 101 of SEQ ID NO: 1 and/or a Thymineat nucleotide 945 of SEQ ID NO: 2 and/or a Thymine at nucleotide 68 ofSEQ ID NO: 3 and/or an Adenine at nucleotide 227 of SEQ ID NO: 4 and/ora Cytosine at nucleotide 839 of SEQ ID NO: 5 and/or an Adenine atnucleotide 445 of SEQ ID NO:
 6. 3. A cultivated melon plant of thespecies Cucumis melo comprising melon plant cells according to claim 1.4. A cultivated melon seed of the species Cucumis melo comprising melonplant cells according to claim
 1. 5. A cultivated melon fruit of thespecies Cucumis melo comprising melon plant cells according to claim 1.6. Melon plant propagation material of the species Cucumis melocomprising melon plant cells according to claim
 1. 7. A method forproducing a ToLCNDV resistant melon plant, comprising: a) selecting aToLCNDV resistant donor plant, b) crossing the donor plant selected ina) with a plant sensitive to ToLCNDV, c) obtaining seeds from the plantscrossed in b), wherein the seeds comprise an introgression fragment fromchromosome 5 of a ToLCNDV resistant donor plant, wherein theintrogression fragment confers ToLCNDV resistance and comprises aThymine at nucleotide 68 of SEQ ID NO: 3 and/or an Adenine at nucleotide227 of SEQ ID NO: 4, and wherein the introgression fragment isobtainable from seeds of which a representative sample has beendeposited under Accession Number NCIMB
 42585. 8. A method for producingmelon seeds, comprising: a) growing a melon plant comprising at leastone chromosome 5 having an introgression fragment from chromosome 5 of aToLCNDV resistant donor plant, wherein the introgression fragmentconfers ToLCNDV resistance and comprises a Thymine at nucleotide 68 ofSEQ ID NO: 3 and/or an Adenine at nucleotide 227 of SEQ ID NO: 4, andwherein the introgression fragment is obtainable from seeds of which arepresentative sample has been deposited under Accession Number NCIMB42585, b) harvesting the fruits of the melon plants grown in a), andoptionally c) collecting the seeds from the fruits obtained in b).
 9. Amethod for producing hybrid melon seeds, comprising: a) crossing a firstinbred melon plant comprising at least one chromosome 5 having anintrogression fragment from chromosome 5 of a ToLCNDV resistant donorplant, wherein the introgression fragment confers ToLCNDV resistance andcomprises a Thymine at nucleotide 68 of SEQ ID NO: 3 and/or an Adenineat nucleotide 227 of SEQ ID NO: 4 with a second inbred melon plant withor without a chromosome 5 having said introgression fragment, andwherein the introgression fragment is obtainable from seeds of which arepresentative sample has been deposited under Accession Number NCIMB42585, and b) selecting seeds obtained from the cross of a).
 10. Amethod for producing a melon fruit, comprising: a) growing a plantcomprising at least one chromosome 5 having an introgression fragmentfrom chromosome 5 of a ToLCNDV resistant donor plant, wherein theintrogression fragment confers ToLCNDV resistance and comprises aThymine at nucleotide 68 of SEQ ID NO: 3 and/or an Adenine at nucleotide227 of SEQ ID NO: 4, and wherein the introgression fragment isobtainable from seeds of which a representative sample has beendeposited under Accession Number NCIMB 42585, and b) harvesting thefruits produced by the plants grown in a).
 11. The cultivated melonplant cell according to claim 1, wherein the introgression fragmentcomprises a Thymine at nucleotide 945 of SEQ ID NO: 2, a Thymine atnucleotide 68 of SEQ ID NO: 3, and an Adenine at nucleotide 227 of SEQID NO:
 4. 12. The cultivated melon plant cell according to claim 1,wherein the introgression fragment comprises a Thymine at nucleotide 945of SEQ ID NO: 2, a Thymine at nucleotide 68 of SEQ ID NO: 3, an Adenineat nucleotide 227 of SEQ ID NO: 4, and a Cytosine at nucleotide 839 ofSEQ ID NO:
 5. 13. The cultivated melon plant cell according to claim 1,wherein the introgression fragment comprises a Thymine at nucleotide 945of SEQ ID NO: 2, a Thymine at nucleotide 68 of SEQ ID NO: 3, an Adenineat nucleotide 227 of SEQ ID NO: 4, a Cytosine at nucleotide 839 of SEQID NO: 5, and an Adenine at nucleotide 445 of SEQ ID NO:
 6. 14. Thecultivated melon plant according to claim 3, wherein the plant is an F1hybrid, comprising the introgression fragment in homozygous orheterozygous form.
 15. The cultivated melon plant according to claim 3,wherein the plant is of the species Cucumis melo cantalupensis, Cucumismelo inodorous or Cucumis melo reticulatus.
 16. The cultivated melonplant according to claim 3, wherein the introgression fragment comprisesa Cytosine at nucleotide 101 of SEQ ID NO: 1, a Thymine at nucleotide945 of SEQ ID NO: 2, a Thymine at nucleotide 68 of SEQ ID NO: 3, aAdenine at nucleotide 227 of SEQ ID NO: 4, a Cytosine at nucleotide 839of SEQ ID NO: 5 and a Adenine at nucleotide 445 of SEQ ID NO: 6, orprogeny thereof retaining the introgression fragment.
 17. The cultivatedmelon seed according to claim 4, wherein the seed is an F1 hybrid seed,comprising the introgression fragment in homozygous or heterozygousform.
 18. The cultivated melon seed according to claim 4, wherein theintrogression fragment comprises a Cytosine at nucleotide 101 of SEQ IDNO: 1, a Thymine at nucleotide 945 of SEQ ID NO: 2, a Thymine atnucleotide 68 of SEQ ID NO: 3, a Adenine at nucleotide 227 of SEQ ID NO:4, a Cytosine at nucleotide 839 of SEQ ID NO: 5 and a Adenine atnucleotide 445 of SEQ ID NO: 6, or progeny thereof retaining theintrogression fragment.
 19. A method of screening a plant or plantmaterial or DNA derived therefrom for the presence of a fragment onchromosome 5 conferring TolCNDV resistance comprising screening genomicDNA from said plant or plant material for the SNP genotype of one ormore of SNP 01 at nucleotide 101 of SEQ ID NO: 1, SNP 02 at nucleotide945 of SEQ ID NO: 2, SNP 03 at nucleotide 68 of SEQ ID NO: 3, SNP 04 atnucleotide 227 of SEQ ID NO: 4, SNP 05 at nucleotide 839 of SEQ ID NO: 5and SNP 06 at nucleotide 445 of SEQ ID NO: 6 and optionally selecting aplant or plant material that comprises the resistant donor genotype ofone or more of SNP 01, SNP 02, SNP 03, SNP 04, SNP 05 and SNP 06 whereinthe resistant donor genotype comprises a Cytosine for SNP 01 atnucleotide 101 of SEQ ID NO: 1, a Thymine for SNP 02 at nucleotide 945of SEQ ID NO: 2, a Thymine for SNP 03 at nucleotide 68 of SEQ ID NO: 3,an Adenine for SNP 04 at nucleotide 227 of SEQ ID NO: 4, a Cytosine forSNP 05 at nucleotide 839 of SEQ ID NO: 5, and an Adenine for SNP 06 atnucleotide 445 of SEQ ID NO: 6, wherein the introgression fragment isobtainable from seeds of which a representative sample has beendeposited under Accession Number NCIMB
 42585. 20. A method foridentifying or detecting a cultivated Cucumis melo plant comprising anintrogression fragment on chromosome 5, wherein said introgressionfragment comprises a ToLCNDV-resistance allele, comprising screening aCucumis melo plant using a molecular marker assay that detects at leastone of SNP marker of SNP 01 at nucleotide 101 in SEQ ID NO: 1, SNP 02 atnucleotide 945 in SEQ ID NO: 2, SNP 03 at nucleotide 68 in SEQ ID NO: 3,SNP 04 at nucleotide 227 in SEQ ID NO: 4, SNP 05 at nucleotide 839 inSEQ ID NO: 5 and/or SNP 06 at nucleotide 445 in SEQ ID NO: 6, andidentifying and/or selecting a plant comprising the CC or CT genotypefor SNP 01 in SEQ ID NO: 1, and/or the TT or TG genotype for SNP 02 inSEQ ID NO: 2, and/or the TT or TC genotype for the SNP 03 in SEQ ID NO:3 and/or the AA or AG genotype for the SNP 04 in SEQ ID NO: 4, and/orthe CC or CT genotype for SNP 05 in SEQ ID NO: 5, and/or the AA or AGgenotype for SNP 06 in SEQ ID NO: 6, wherein the introgression fragmentis obtainable from seeds of which a representative sample has beendeposited under Accession Number NCIMB
 42585. 21. The cultivated melonplant cell according to claim 1, wherein the introgression fragmentcomprises the sequence of the ToLCNDV resistant donor plant betweenSNP_03 at nucleotide 68 of SEQ ID NO: 3 and SNP_04 at nucleotide 227 ofSEQ ID NO:
 4. 22. A cultivated melon plant cell of the species Cucumismelo comprising an introgression fragment from chromosome 5 of a ToLCNDVresistant donor plant, wherein the introgression fragment confersToLCNDV resistance and wherein the introgression fragment comprises thesequence of the ToLCNDV resistant donor plant between SNP_03 atnucleotide 68 of SEQ ID NO: 3 and SNP_04 at nucleotide 227 of SEQ ID NO:4, and wherein the introgression fragment is obtainable from seeds ofwhich a representative sample has been deposited under Accession NumberNCIMB
 42585. 23. The method of claim 7, further comprising d) verifyingif the plants grown from the seeds obtained in c) are resistant toToLCNDV and/or comprise one or more of the SNP nucleotides from thedonor plant for SNP_01, SNP_02, SNP_03, SNP_04, SNP_05 or SNP_06,wherein the SNP nucleotide from the donor plant is a Cytosine atnucleotide 101 of SEQ ID NO: 1 for SNP_01, a Thymine at nucleotide 945of SEQ ID NO: 2 for SNP_02, a Thymine at nucleotide 68 of SEQ ID NO: 3for SNP_03, an Adenine at nucleotide 227 of SEQ ID NO: 4 for SNP_04, aCytosine at nucleotide 839 of SEQ ID NO: 5 for SNP_05, and an Adenine atnucleotide 445 of SEQ ID NO: 6 for SNP_06.